PHARMACEUTICAL MICROBIOLOGY - V. A. Galynkin - 2015

PART I. MICROBIAL BIOLOGY

CHAPTER I. PROKARYOTES (BACTERIA)

1.1 Structure OF THE Introduction/4.html">Prokaryotic Cell

All Living organisms are divided into three major realms of existence: the animal kingdom, the plant kingdom, and the protist world. Our planet is home to up to 3 million species of animals and about half a million species of plants. In 1886, the German biologist E. Haeckel proposed grouping all single-celled microorganisms lacking Organ and tissue differentiation (Protozoa, Fungi, Bacteria) into a separate kingdom, Protista, which includes organisms that in many respects occupy an intermediate position between plants and animals. Later, based on Cellular Organization, protists were subdivided into two distinct groups: lower organisms (prokaryotes) (Fig. 1) and higher organisms (eukaryotes) (Fig. 2).

Class="center">Fig. 1. Diagram of The structure of a prokaryotic cell [1].

Fig. 2. Model of Eukaryotic Cell organization [1].

The size of most Prokaryotic Cells ranges from 0,2 to 10,0 µm (Table 1). The largest prokaryotes isolated to date are cells of Epulopiscium fishelsoni, which inhabit the gut of deep-sea surgeonfish, reaching up to 600 µm in length and up to 100 µm in diameter, and cells of Thiomargarita namibiensis, found in the coastal waters of Chile and Namibia, ranging from 400 to 750 µm in diameter. At the same time, There is a very small marine alga, Nanochlorum eukaryotum, which nevertheless possesses a true nucleus, METABOLISM/14.html">Chloroplasts, and Mitochondria. Summarizing these data, we can conclude that the sizes of currently known prokaryotic microorganisms range from 0,05 to 750 µm.

Table 1. Dimensions of selected Representatives of the living world

Lower protists include those whose cellular structure differs significantly from all other organisms (bacteria and blue-green Algae); these are the prokaryotes (pre-nuclear cells).

In higher protists, cells resemble PLANT AND ANIMAL cells; these are eukaryotes, i.e., microorganisms possessing a true nucleus (from the Greek eu meaning true, and karyon meaning kernel or nucleus). The Nucleus is separated from the surrounding Cytoplasm by a double-layered nuclear membrane containing pores. Inside the nucleus are 1 to 2 nucleoli, which are centers of ribosomal RNA Synthesis, and Chromosomes—the primary carriers of hereditary information composed of DNA and protein. During Cell Division, chromosomes are distributed between daughter cells through complex processes known as Mitosis and Meiosis. The eukaryotic cytoplasm contains mitochondria, and in photosynthetic organisms, chloroplasts. The cytoplasmic membrane surrounding The Cell extends into the cytoplasm to form The Endoplasmic reticulum; there is also a membranous organelle, the Golgi apparatus, as a cytoplasmic component (Fig. 2).

Eukaryotes include microscopic algae (excluding blue-green algae) and microscopic fungi (Molds and Yeasts).

Bacteria have colonized A wide variety of habitats: they live in soil, dust, Water, air, on the exterior surfaces of plants and animals, and inside host organisms. They can even be found in hot springs, surviving at temperatures around 60°C or higher. It is difficult to determine the exact population of bacteria: 1 g of fertile soil can contain up to 100 million bacteria, while 1 cm³ of fresh milk may harbor 3,000. Microbial activity is of vital importance to all other living creatures, as bacteria and fungi break down organic matter and participate in the global cycling of elements. Furthermore, bacteria are playing an increasingly significant role in human life, not merely because they cause various diseases, but because they can be harnessed to produce many essential products.

Prokaryotic cells have a simpler organization than Eukaryotic cells. They lack a distinct boundary between the nucleus and cytoplasm and do not possess a nuclear membrane; their DNA does not form structures resembling eukaryotic chromosomes, and consequently, prokaryotes undergo only simple division rather than classical mitosis. Most prokaryotes lack membrane-bound intracellular Organelles, as well as Mitochondria and chloroplasts; Ribosomes lie freely in the cytoplasm.

Prokaryotic organisms include blue-green algae, bacteria, rickettsiae, actinomycetes, and mycoplasmas [2, 3].

To date, more than 3,500 species of bacteria have been described, but this number is constantly growing. Making sense of this staggering diversity is possible thanks to systematics. Systematics (from the Greek systema meaning a whole compounded of parts, and systematicus meaning ordered) is the science dedicated to the Classification of organisms, their evolutionary relationships, and interconnections.

Classification (from the Latin classis meaning rank or group) is the arrangement of a set of organisms into classes and groups (taxons) based on their shared characteristics; it is a core component of systematics.

Taxonomy (from the Greek taxis meaning arrangement or order, and nomos meaning law) is the theoretical framework of Biological Classification and systematics.

The terms "systematics" and "taxonomy" are often used interchangeably, although systematics is the broader concept.

Systematics comprises three independent components: classification, identification, and nomenclature. Classification, as already mentioned, is the sorting of organisms into taxonomic groups.

Identification is The process of determining whether an organism under study belongs to a particular taxon (class, order, family, genus, species, etc.).

Nomenclature is a set of rules governing the naming of taxa, along with the list of these names. Nomenclature represents the concluding stage of systematics following classification; it functions as an "information language" and is, to a certain extent, independent of classification.

Until the second half of the 19th century, biological classification was based entirely on the external, observable traits of organisms—their phenotypes (such as Morphology, motility, Gram-staining characteristics, presence of capsules, endospore formation, cultural and biochemical properties, and other markers)—because the hereditary material, the genotype, was still beyond the reach of scientific investigation.

Consequently, traditional or classical taxonomy, which relies on studying external features expressed during the organism's life cycle, is purely phenotypic classification (phenetics). As more phenotypic data became accessible to researchers and computers were adopted for data Processing, a new approach emerged: numerical taxonomy (or phenetic numerics).

The advent of molecular biology in the 1950s and its rapid advancement paved the way for a new branch of taxonomy known in Eastern European literature as genosystematics. Unlike phenetics, which examines a multitude of phenotypic traits, genosystematics focuses on a single molecule—the cellular genetic material (DNA) that programs the organism's individual development. In other words, genosystematics is a branch of taxonomy dedicated to studying genotypes, the genetic blueprints shaped by biological evolution on Earth. The fundamental difference between phenetics and genosystematics lies in their core Objects of Study.

When classifying related microorganisms, the following taxonomic categories are used: kingdom (regnum), division (divisio), section (section), class (classis), order (ordo), family (familia), genus (genus), and species (species). Microorganisms are named in strict accordance with the rules of the International Code of Nomenclature of Bacteria.

In microbiology, as in general biology, bacterial species are designated using the binomial (binary) nomenclature system proposed back in the 18th century by Carl Linnaeus. According to this system, the genus name is written with a capital initial letter and appears first; it indicates the microbial genus (which may reflect a morphological trait, honor the scientist who discovered the microbe, etc.). The second word, written with a lowercase letter, denotes the species. Typically, a species epithet is derived from a noun describing the color of the colony, the organism's habitat, the disease or process it causes, or other distinguishing characteristics. For example, Escherichia coli indicates that the microbe was discovered by Escherich, and coli refers to its habitation in the colon; Bacillus anthracis indicates a spore-forming rod, with anthracis denoting the CAUSATIVE AGENT OF anthrax; and Azotobacter refers to an atmospheric nitrogen-fixing microorganism.

The fundamental unit of nomenclature is the species. V. D. Timakov (1973) defined species as follows: "A species is a population of microorganisms sharing a common origin and genotype, possessing similar morphological and biological properties, and having a hereditarily fixed capacity to elicit qualitatively specific processes in their natural habitat." Species are further subdivided into subspecies or variants. If isolated bacteria exhibit deviations from typical species characteristics, the culture is classified as a subspecies. There are also infrasubspecies categories based on minor hereditary variations: antigenic variants (serovars), biochemical variants (biovars), phage-susceptibility variants (phagovars), and pathogenicity variants (pathovars), among others. Incorporating the suffix "-var" (variant) into these terms is recommended to avoid confusion, whereas the previously used term "type" is now reserved strictly for nomenclatural types.

Microbiology makes frequent use of terms such as "pure culture," "mixed culture," "clone," and "strain." A "culture" refers to microorganisms grown in a laboratory on solid or in liquid nutrient media. A microbial culture consisting of individuals of a single species is called a pure culture. A mixed culture is a mixture of heterogeneous organisms that have grown together in a nutrient medium either upon initial inoculation of a sample (such as milk, soil, water, or clinical material) or through environmental contamination of a medium previously seeded with a single microbial species. A clone is a culture derived from a single cell population of a specific microbial species. A strain is a pure culture of a specific microbial species isolated from a particular source that differs from the reference strain in minor properties (such as antibiotic sensitivity or carbohydrate Fermentation patterns).

Consequently, prokaryotes constitute a distinct taxonomic group of microorganisms that differ substantially from eukaryotic microorganisms, which include fungi, plants, animals, and humans (Table 2). In scientific literature, prokaryotes are traditionally referred to simply as "bacteria" [4]. Depending on Cell wall characteristics, bacteria are divided into four primary categories: (1) Gram-negative bacteria, (2) Gram-positive bacteria, (3) cell wall-less bacteria, and (4) archaea. The first two groups possess rigid cell walls whose structural framework consists of peptidoglycan (murein) containing muramic acid. This group encompasses the majority of human pathogens and saprotrophic microorganisms. Bacteria lacking a rigid cell wall—and consequently devoid of a fixed cellular shape—are called mycoplasmas. This group includes species pathogenic to humans (Mycoplasma pneumoniae, M. hominis, M. fermentans), animals, and plants.

Archaea lack murein in their cell walls, rendering them naturally resistant to β-lactam Antibiotics. They differ significantly from other prokaryotes in their molecular architecture. Primarily soil or aquatic microorganisms, they typically inhabit extreme environments (such as hypersaline, highly acidic, or high-Temperature habitats). In addition, some archaea exist as symbionts in the digestive tracts of warm-blooded animals.

1.2 Morphology

Prokaryotes are unicellular microorganisms whose cells typically range from 0.2 to 2 µm in diameter (Fig. 1). Based on cell shape, they are divided into three main groups: spherical (cocci), cylindrical (bacteria, bacilli), and spiral (spirilla, spirochetes) (Fig. 3).

Fig. 3. Representatives of the prokaryotic world.

Table 2. Morphology of PROKARYOTES AND EUKARYOTES

Based on cell shape, they are divided into three main groups: spherical (cocci), cylindrical (rods, bacilli), and spiral (spirilla, spirochetes) (Fig. 4). Cocci can vary in shape from spherical to ellipsoidal, Kidney-shaped (bean-shaped), and lanceolate. Depending on their cellular arrangement, they are classified into

diplococci, streptococci, tetrads, sarcinae, and staphylococci.

Fig. 4. Bacterial morphology. A. Spherical cells grouped in clusters (1 — Staphylococcus), pairs (2, 3 — Neisseria), chains (4 — Streptococcus), tetrads (5 — Micrococcus), and cubic packets (6 — Sarcina). B. Cylindrical (rod-shaped) cells. C. Vibrios. D. Spirilla. E. Spirochete — structural diagram: a — outer membrane, b — protoplasmic cylinder, c — periplasmic flagella [2].

Micrococci (Micrococcus) are characterized by single, paired, or haphazard cellular arrangements. This genus includes opportunistic pathogens (M. luteus) as well as saprotrophic species.

Diplococci occur in pairs. This group includes the causative agents of meningitis and Gonorrhea.

Streptococci (from streptos, meaning twisted) are arranged in chains. This group includes pathogenic species (such as S. pyogenes).

Tetrads are arranged in groups of four, whereas sarcinae (from sarcio, meaning to join) form cubical packets of 8, 16, or more cells. These are predominantly saprotrophic species.

Staphylococci (from staphylos, meaning a bunch of grapes) form clusters resembling grapes. This group includes pathogenic species such as S. aureus.

Cylindrical (rod-shaped) microorganisms include non-spore-forming rods (bacteria), bacilli, and clostridia (spore-forming forms). They vary in size, shape, and cellular arrangement. This group encompasses members of the normal human microbiota (such as Escherichia coli) alongside various pathogens (including the causative agents of intestinal infections, tetanus, anthrax, and anaerobic infections).

Spiral forms include spirilla, which have a rigid cell wall, and spirochetes, whose cell wall is elastic, providing motility. Spirillum minor is the causative agent of sodoku, a disease transmitted through rodent bites. Spirochetes are responsible for many infectious diseases (see below).

In addition to these main forms, some prokaryotes feature a more complex structure. These are actinomycetes, which include the majority of antibiotic producers as well as several pathogenic species.

1.3 Actinomycetes

Actinomycetes are classified as Gram-positive bacteria. They share similarities with bacteria in their lack of a true nucleus (prokaryotes) and are classified as mycelial prokaryotes. However, based on their morphological, physiological, biological, and ecological characteristics, actinomycetes form a distinct, independent group. The vegetative body of actinomycetes (mycelium) consists of very thin branching hyphae (5–7 times thinner than fungal hyphae) (Fig. 5).

Fig. 5. Actinomycetes. Growth diagram of aerial and substrate mycelium in the genera: 1 — Nocardia, 2 — Actinoplanes, 3-5 — Streptomyces, 6 — Streptoverticillium [2].

Actinomycetes reproduce through mycelial fragments or spores. On nutrient media, they initially form leather-like colonies (substrate mycelium), which are subsequently covered by an aerial mycelium. The colony itself grows into the Agar via the substrate mycelium. Actinomycetes are not specialized in their Nutrition, utilizing various plant and animal residues.

Most actinomycetes are saprotrophic, with only a few having adapted to a parasitic lifestyle on plants.

Among pathogenic actinomycetes, species of the genus Streptomyces, which cause plant scab, are of greatest interest. The most well-known are common scab in potato tubers and scab in ROOT crops such as beets and carrots.

Potato scab is caused by Streptomyces Scabies. The disease develops on tubers during the plant's growing season. Cracks and small warts appear at the infection sites, the affected tissue undergoes suberization, and ulcers are formed.

Oxygenic phototrophic bacteria (cyanobacteria) inhabit aquatic and terrestrial environments and cause water blooms; some species produce toxins that pose hazards when consuming bloom-affected water. They can serve as a source of BIOLOGICALLY ACTIVE SUBSTANCES (phycocyanin) or be consumed as a dietary Supplement (Spirulina maxima).

In microbiology, There are two distinct approaches to taxonomy that give rise to Two Types of classification. The first is based on establishing a natural (phylogenetic) classification of prokaryotes—that is, constructing a unified system that objectively reflects the evolutionary relationships among various microbial groups and their historical development. The second taxonomic approach pursues practical goals and serves for identification, which is determining whether a microorganism belongs to a specific species. This is an artificial (traditional) classification. Modern microorganism classification systems are essentially all artificial. They serve as the basis for diagnostic keys used to identify specific microorganisms, such as N. A. Krasilnikov's «Key to Bacteria and Actinomycetes» (1949).

1.4 Classification of Prokaryotic Microorganisms

In D. H. Bergey's Manual of Determinative Bacteriology [3], all prokaryotic microorganisms are united into the kingdom Procaryotae, which is subdivided into four divisions, further divided into sections, classes, orders, families, genera, and species.

Division I. Gracilicutes (from Lat. gracilis — thin, slender, cutis — Skin). Includes Gram-negative microorganisms. The division comprises nine sections.

Section 1. Spirochetes. Order Spirochaetales. Includes two families: Spirochaetaceae (four genera) and Leptospiraceae (one genus).

Section 2. Spiral and curved aerobes (microaerophiles). A single family, Spirillaceae, containing six genera. Microorganisms of the genus Campylobacter are pathogenic to humans and animals.

Section 3. Gram-negative non-motile curved bacteria. A single family, Spirosomonaceae, which includes three pathogenic genera.

Section 4. Aerobic Gram-negative rods and cocci. Eight families, two of which contain pathogenic microorganisms. The family Pseudomonadaceae includes four genera and over 25 species, some of which are pathogenic (A. mallei, etc.). The family Neisseriaceae comprises 16 genera. The genera Neisseria and Moraxella contain microorganisms pathogenic to humans and animals.

The genera Bordetella, Brucella, and Francisella are not assigned to families and contain microorganisms pathogenic to humans and animals.

Section 5. Gram-negative facultative anaerobes. Three families: Enterobacteriaceae, Vibrionaceae, and Pasteurellaceae. The family Enterobacteriaceae comprises 14 genera (Escherichia, Salmonella, Citrobacter, Klebsiella, Enterobacter, Erwinia, Shigella, Proteus, Yersinia, etc.). The family Vibrionaceae has two genera. The genus Vibrio includes pathogenic microorganisms. The family Pasteurellaceae has three main genera: Pasteurella, Haemophilus, and Actinobacillus. These contain pathogenic microbial species.

Section 6. Strict anaerobes. Curved Gram-negative rods. A single family, Bacteroidaceae, containing 13 genera, some of which are pathogenic.

Section 7. Dissimilatory sulfate-reducing bacteria. Seven non-pathogenic genera.

Section 8. Anaerobic Gram-negative cocci. A single family, Veillonellaceae, containing three genera.

Section 9. Rickettsiae and chlamydiae. Two orders: Rickettsiales and Chlamydiales. The order Rickettsiales includes three families: Rickettsiaceae, Bartonellaceae, and Anaplasmataceae. The family Rickettsiaceae has three tribes comprising eight genera. The family Bartonellaceae contains two genera, and Anaplasmataceae contains four. The order Chlamydiales has one family, Chlamydiaceae, and one genus, Chlamydia. All families contain pathogenic microorganisms.

Division II. Firmicutes (from Lat. firmis — strong, cutes — skin). This division mainly comprises Gram-positive bacteria.

Section 12. Gram-Positive Cocci. Two families: Micrococcaceae and Deinococcaceae. The family Micrococcaceae includes four genera: Micrococcus, Stomatococcus, Planococcus, and Staphylococcus.

In addition to these two families, the section includes ten independent genera: Streptococcus, Leuconostoc, Pediococcus, Sarcina, etc.

Section 13. Spore-Forming Gram-Positive Rods and Cocci. Six genera: Bacillus, Clostridium, Sporolactobacillus, Sporosarcina, etc. The first two genera contain pathogenic species.

Section 14. Non-Spore-Forming Gram-Positive Rods. Seven genera: Lactobacillus, Listeria, Erysipelothrix, etc. Pathogenic species are present.

Section 15. Non-Spore-Forming Intracellular Gram-Positive Rods. 21 genera:

Corynebacterium, Mycobacterium, Propionibacterium, Eubacterium, Azotobacterium, Bifidobacterium, Actinomyces, etc.

Section 16. Mycobacteria. One family: Mycobacteriaceae. The family comprises a single genus, Mycobacterium, which includes 49 species: Myc. tuberculosis, Myc. bovis, Myc. avium, Myc. paratuberculosis, Myc. leprae, etc.

Section 17. Nocardioforms. Nine genera: Nocardia, Pseudococcus, Pseudonocardia, etc.

Division III. Tenericutes. This groups together Gram-negative prokaryotes lacking a cell wall but possessing a cytoplasmic membrane. The tenth section in this division comprises the mycoplasmas of the class Mollicutes (from Lat. molli — soft, cutes — covering, skin). The class contains a single order, Mycoplasmatales, and three families: Mycoplasmataceae, Acholeplasmataceae, and Spiroplasmataceae. Pathogenic mycoplasmas are primarily included in the family Mycoplasmataceae.

Section 11. Endosymbionts.

Division IV. Mendosicutes. Prokaryotes that include no pathogenic bacteria; these are methane-producing, sulfur-oxidizing, halophilic, mycoplasma-like, thermoacidophilic, and other evolutionarily most ancient bacteria (archaea).

1.5 Cell Structure

The cell wall maintains the structural rigidity of the cell, preserves its constant shape, and provides mechanical strength. In addition, it acts as an osmotic barrier featuring selectively permeable zones for substances of various chemical natures. It contains the peptidoglycan murein as a supportive framework. The backbone of murein (Fig. 6) consists of chains of alternating N-acetylglucosamine and N-acetylmuramic acid residues linked by β-1,4-glycosidic bonds. The muramic acid residues are cross-linked by polypeptide chains composed of α-Alanine, D-alanine, Lysine, D-glutamic acid, and meso-diaminopimelic acid.

Fig. 6. Peptidoglycan of E. coli. A — N-acetylmuramic acid; B — N-acetylglucosamine; ala — alanine; glu — glutamic acid; DAP — diaminopimelic acid.

D-Amino Acids and muramic acid are unique to prokaryotes, and the distinct structure of the cell wall serves as the basis for the selective Action of Certain antibiotics, such as penicillin and other β-lactams. The cell walls of Gram-positive and Gram-negative bacteria differ substantially in their structure.

In Gram-negative bacteria, the murein network is single-layered, sometimes double-layered, and accounts for no more than 10% of the dry weight of the cell wall. Proteins, Lipoproteins, lipopolysaccharides, and Phospholipids forming the outer membrane are located on it (Fig. 7). The stabilization of these components is ensured by Ca²⁺ and Mg²⁺ ions. Lipid A is of critical importance for the Structure and function of the outer membrane. Its backbone contains a disaccharide composed of D-glucosamine residues linked by a β-1,6-bond, with phosphate groups at positions 1 and 4. The backbone is esterified with C₁₂, C₁₄, and C₁₆ fatty acids. Lipid A possesses a unique conformation—compact and highly ordered—thereby creating a viscous structure within the membrane that impedes the diffusion of Bile acids, detergents, and certain antibiotics. Lipid A provides the toxicity and pyrogenicity of the lipopolysaccharide. The antigenic Specificity of Gram-negative bacteria is primarily determined by the CARBOHYDRATES of the O-specific side chains protruding outward from the cell surface. The outer membrane confers a high resistance to antimicrobial agents in Gram-negative bacteria compared to Gram-positive ones.

Fig. 7. Structural model of the cytoplasmic membrane in Gram-negative bacteria: The lipopolysaccharide molecule is shown on the right. Glu — glucose; Glu-N — glucosamine; NA = N-AcGlu — N-acetylglucosamine; Gal — galactose; Hep — heptose; KDO — 2-keto-3-deoxyoctonic acid; M — murein; OM — outer membrane; PM — Plasma Membrane; PP — periplasmic space [7].

Broad-spectrum antibiotics, such as beta-lactams, cause the release of lipopolysaccharides from the outer membrane, which can lead to endotoxic Shock in the patient.

In Gram-positive bacteria, the outer membrane is absent, while the murein network constitutes 30–70% of the cell wall dry weight and reaches up to 40 layers. The presence of teichoic and teichuronic acids (Fig. 8) is characteristic, which impart a negative charge to the cell and facilitate the sorption of cations from the environment. Additional components—such as Lipids, Waxes, mycolic acids, proteins, and Polysaccharides—may be present in some microorganisms.

Fig. 8. Structural model of the cytoplasmic membrane of Gram-positive bacteria. A — glycerol teichoic acid; B — ribitol teichoic acid; G — glycosyl; Ala — alanyl.

Differences in the cell wall structure of the two groups of microorganisms are revealed using Gram staining. The specimen is treated with a solution of crystal violet and then iodine. The resulting dye-iodine complex is localized on the protoplast. When the specimen is treated with alcohol, it is retained by the cell wall of Gram-positive bacteria and washed out from Gram-negative ones. The ability to undergo Gram staining is an important taxonomic trait that correlates with other bacterial properties.

The S-layer (from surface) is located On the surface of all prokaryotic cells, covering the entire cell. It consists of structural units—proteins or Glycoproteins forming a monolayer whose structure is typical of two-dimensional crystals (having hexagonal, oblique, or square Symmetry lattices). The interaction between subunits and underlying structures occurs via non-covalent bonds. The S-layer protects the cell against external influences; however, during prolonged cultivation, it can be lost without compromising the viability of the strain.

Capsules and slime are formed by certain bacteria outside the cell wall as its outer layer. The ability to form them is not a species-specific trait, as both encapsulated and non-encapsulated strains can exist. In pathogenic microorganisms, the capsule provides protection against phagocytosis, thereby increasing strain virulence (as in pneumococci). In soil- and plant-dwelling microbes, the capsule protects cells from desiccation, solar radiation, and biocides. Capsules and slime create osmotic conditions for microbial cells that favor the sorption of nutrients from the substrate and promote cell-cell and cell-substrate adhesion. Many exoenzymes are localized within the capsule, where transformations of substances entering the cell take place (Fig. 9).

Fig. 9. Capsules surrounding Clostridium cells.

In most bacteria, capsules and slime are of a polysaccharide nature. In certain bacilli, they are Polypeptides composed mainly of D- and L-glutamic acid.

Capsular polysaccharides possess antigenic specificity and are used in vaccine manufacturing (for pneumococci and meningococci) as well as for identification and classification (in salmonellae). Soluble slimes (such as dextran from Leuconostoc dextranicum and L. mesenteroides, or xanthan from Xanthomonas campestris) are produced on an industrial scale and widely used in pharmacy and other fields.

Protoplasts and spheroplasts are structures that have completely (protoplasts) or partially (spheroplasts) lost their cell wall, for instance, under the action of Lysozyme or penicillin. These are osmotically labile elements that can exist only in hypertonic solutions. They retain biological activity and are capable of reverting to normal cells under special conditions. They are utilized in cell engineering to obtain hybrid microorganism forms.

L-forms, named after the Lister Institute in London, are formed under conditions that disrupt cell wall synthesis; for example, in a patient with tuberculosis, the causative agent may transform into an L-form under The Influence of medications. In this process, mycobacteria lose their characteristic acid-fastness, which complicates their detection and disease Diagnosis. Such cells are characterized by irregular, sometimes filamentous shapes capable of passing through bacterial filter pores. Labile L-forms can revert to normal cells. Stable forms do not form a cell wall because its loss is associated with a genotype alteration (mutation).

The periplasmic space is located between the murein layer and the cytoplasmic membrane. It contains hydrolase enzymes that break down substances entering the cell, polymerases involved in cell wall and capsule synthesis, proteins participating in substrate transport into the cytoplasm, and receptor proteins for chemotactic stimuli.

The cytoplasmic membrane lies beneath the cell wall and separates the cytoplasm from the external environment. It has a thickness of 6-8 nm and accounts for 8-15% of the cell's dry weight. Its structure conforms to the general organizational principle of prokaryotic and eukaryotic cell membranes. It consists of two layers of lipid molecules, in which the hydrophobic fatty acid chains are oriented perpendicularly to its plane, while the hydrophilic polar PARTS OF THE molecules are linked to protein molecules via polar, electrostatic, and hydrophobic interactions. Lipid and protein molecules do not maintain a fixed orientation but are in constant motion within the framework of this membrane (Fig. 10).

Fig. 10. Structure of the Bacterial cell wall. Top: Gram-positive cell wall. 1. Cytoplasmic membrane, 2 — PG, 3 — phospholipid, 4 — protein, 5 — lipoteichoic acid. Bottom: Gram-negative cell wall. 1 — inner membrane, 2 — periplasmic space, 3 — outer membrane, 4 — phospholipid, 5 — PG, 6 — LP, 7 — protein, 8 — LPS, 9 — porins.

Structure of the Gram-positive cell wall. The cell wall contains up to 40 layers of peptidoglycan. Teichoic acid molecules are covalently linked to peptidoglycan (PG). Lipoteichoic acids (LTAs) contain lipid "tails" anchored in the hydrophobic region of the CPM. Cell walls may feature surface protein layers. Protein structures are either arranged in patches (as shown in the diagram) or densely packed to form an S-layer.

Structure of the Gram-negative cell wall. A thin layer of peptidoglycan is overlaid by an outer membrane (OM), attached to it by lipoproteins (LPs). Between the two membranes lies the periplasmic space containing a peptidoglycan (PG) layer. The inner surface of the outer membrane contains phospholipids, while the outer surface contains lipopolysaccharides (LPS). LPS molecules consist of a lipid portion facing inward toward the outer membrane to form its hydrophobic region, and a polysaccharide portion facing the external environment.

Lipids, predominantly polar phospholipids, make up about 40% of the membrane mass. They perform structural functions and ensure the conformational changes of enzyme molecules required for their activity expression.

Membrane Proteins are subdivided into peripheral and integral. The former are located on the surface, whereas the latter are embedded within the membrane thickness. Porin proteins line the membrane pores, ensuring its permeability. Certain membrane proteins perform catalytic functions or act as receptors binding substances essential for the cell.

The cytoplasmic membrane serves as an osmotic barrier, localizing The Active Transport systems for moving substances into and out of the cell.

Mesosomes are specialized structures formed by membrane invagination (folding inward); they contain the Enzymes of the Oxidative Phosphorylation system and function as mitochondria in prokaryotes.

Cytoplasm constitutes the internal environment of the cell. It is a complex, highly heterogeneous system housing the cell's genetic material (nucleoid, Plasmids), 70S ribosomes, metabolic enzyme systems, and reserve substances (polysaccharides, lipids, poly-beta-hydroxybutyric acid, polyphosphates, and sulfur in sulfur bacteria). The majority of metabolic processes take place in the cytoplasm. Membrane structures located within the cytoplasm are most developed in eukaryotic cells, whereas bacteria possess analogues thereof (mesosomes, vacuoles, lysosomes).

Flagella are designed for bacterial locomotion (Fig. 11).

Fig. 11. Organelles of motility in microorganisms: A — Tetrahymena thermophila; B — structure of a bacterial flagellum.

Cyanobacteria, gliding bacteria, and spirochetes are capable of moving without flagella. The number of flagella and their arrangement on the cell constitute a taxonomic feature characteristic of specific species.

Monotrichates are bacteria with a single flagellum at one pole (Vibrio cholerae); amphitrichates have polar flagella at both ends (Spirillum volutans); lophotrichates feature a tuft of flagella at one end (Alcaligenes faecalis); whereas peritrichates have flagella distributed all over the cell surface (E. coli, Salmonella spp.) (Fig. 12).

Fig. 12. MAIN TYPES OF bacterial flagellation [4]

Bacterial flagella are proteinaceous structures composed of flagellin, measuring 10-20 nm in diameter and up to 20 µm in length. A flagellum is anchored in the cytoplasmic membrane and cell wall by a basal body, which consists of a central rod and two pairs of rings (in Gram-negative bacteria) or a single pair of rings (in Gram-positive bacteria). Flagella rotate driven by a flux of charged particles (Н+, ОН^-, Nа⁺) passing through the rings, powered by the Membrane Potential across the cell. Flagellar activity is regulated by environmental sensing systems, enabling cells to migrate toward optimal conditions (taxis). Approximately 35 genes are involved in the assembly and function of the basal body, alongside more than 20 genes that dictate the direction of movement in response to external stimuli. The entire system is governed by a master Operon regulated by the cAMP-activator protein complex.

Fimbriae (pili or microfibrils) are located on The surface of many bacterial cells, which may harbor up to 10,000 such appendages. They are 3-25 nm in diameter and approximately 12 µm long. Fimbriae mediate Cell-to-Cell adhesion during processes like conjugation, as well as attachment to substrates. Their structural proteins act as receptors for biological recognition. Some fimbriae function as capillaries connected to mesosomes and are involved in Water-Salt Metabolism.

F-pili (sex pili) are structurally similar to fimbriae, but cells typically possess no more than one or two of them. They are found exclusively on cells capable of transferring genetic material via conjugation, functioning as receptors and binding structures in this process. Additionally, they serve as receptors for specific phages.

Prokaryotic spores are specialized structures designed for survival under unfavorable environmental conditions. Compared to vegetative cells, spores exhibit significantly higher resistance to high temperatures, radiation, and chemical agents. They typically form within the bacterial cell when nutrient depletion occurs in the culture medium alongside the accumulation of Metabolic waste products. The biochemical trigger for sporulation is a decrease in the intracellular concentration of guanine NUCLEOTIDES, specifically GTP and GDP. Sporulation is also density-dependent: spores do not form at low cell concentrations. However, sporulation can be restored by adding a filtrate from the late exponential phase of a culture in which cells have already begun to sporulate (see quorum sensing). Spore formation requires DNA Synthesis, as the blockade of Replication halts the expression of previously induced sporulation genes. The process begins with the accumulation of protein reserves at the expense of stored nutrients. Spore proteins contain a significantly higher proportion of Cysteine than vegetative proteins; numerous Disulfide Bonds within these proteins are believed to confer high mechanical strength to the spore coats. During early sporulation, specialized proteins bind to DNA and slightly unwind it, altering the geometry of pyrimidine bases and increasing their resistance to ultraviolet radiation. This stage also sees the synthesis of a characteristic compound, dipicolinic acid (pyridine-2,6-dicarboxylic acid), which is incorporated into the spore coat as a calcium salt.

Morphological Changes in the cell during sporulation (Fig. 13) begin with The formation of anasymmetrically positioned septum. Similar to a conventional division septum, it consists of two membranes with a thin layer of peptidoglycan sandwiched between them. The larger compartment becomes the mother cell, while the smaller one forms the forespore containing the nucleoid. Subsequently, the septum degrades, and the mother cell membrane engulfs the forespore, endowing it with a double membrane. A peptidoglycan layer forms between these membranes to constitute the spore cortex. The next stage involves the assembly of the protein spore coat. Upon maturation, the mother cell lyses. A mature spore contains minimal free water and an elevated lipid content compared to vegetative cells, with its coats accounting for up to 50% of its dry weight. All these adaptations ensure its resilience against environmental stress factors.

Sporulation is characteristic primarily of rod-shaped microorganisms (bacilli and clostridia), including the causative agents of anthrax, tetanus, anaerobic infections, botulism, and certain saprotrophic species. In addition, spores are formed by species of the genera Thermoactinomyces, Sporosarcina, and Sporomusa. All of these, with the exception of the last genus, are Gram-positive.

A spore is a dormant life stage. Under favorable conditions, spores germinate: they swell by absorbing water, their metabolic activity increases, dipicolinic acid is released, and their environmental resistance is lost. Finally, the spore coat ruptures, and a vegetative cell emerges (Fig. 13).

Fig. 13. Developmental cycle of Bacillus subtilis: 1 — vegetative cell; 2 — Formation of the sporulation septum; 3-4 — formation of the spore double membrane; 5 — cortex formation (a); 6 — formation of the protein coat (b); 7 — mature spore; 8 — spore germination [2].

The capacity of microorganisms for sporulation is a critical factor when selecting disinfection and sterilization protocols, given the exceptional resistance of spores to biocidal agents. The most resilient species are employed as biological indicators to evaluate sterilization efficacy: Bac. stearothermophilus for pressurized steam, certain Bac. subtilis strains for dry heat, and Bac. pumilus for ionizing radiation.

Prokaryotic cells (from the Greek pro – before, and karyon – nucleus) lack a true membrane-bound nucleus. In other words, their genetic material (DNA) resides directly in the cytoplasm without being enclosed by a nuclear envelope.

1.6 Genetic Apparatus

The bacterial chromosome is a closed circular DNA molecule approximately 1.3 m in length.

Bacterial DNA is represented by single circular molecules roughly 1 mm long. Each such molecule consists of approximately 5 × 10⁶ Base Pairs. The total DNA content (genome) of a bacterial cell is much smaller than that of a eukaryotic cell, and consequently, the volume of encoded information is also smaller. On average, such a DNA molecule contains several thousand genes, which is roughly 500 times less than in a human cell.

Plasmids and episomes

Plasmids and episomes are small fragments of DNA distinct from the bulk of the cellular genome. They often replicate synchronously with the host DNA but are not essential for cell viability.

Initially, a distinction was made between episomes and plasmids, wherein episomes could integrate into the host DNA, while plasmids could not. Episomes included F-factors and temperate phages. Today, both groups are unified under the general term "plasmids." Plasmids are widespread in nature, and in recent years they have been regarded as intracellular parasites or symbionts even simpler than Viruses. While the debate over whether viruses constitute living organisms will be discussed later, the case of plasmids is even more complex, as they consist solely of DNA molecules. Plasmids regulate their own replication and copy number within the cell, which typically ranges from 1 to 100 depending on the plasmid type. A hallmark of plasmids is incompatibility—the inability of closely related plasmids to coexist stably within the same cell, which serves as the basis for their classification. The primary taxonomic unit for plasmids is the Inc group (incompatibility group). Plasmids belonging to the same Inc group share numerous common properties, including similar molecular weight, a high degree of DNA Homology, and the control of comparable phenotypic traits.

The Mechanism of incompatibility involves the inhibition of replication of an incoming plasmid by a specific protein or RNA molecule produced under the control of the resident plasmid already present in the cell. Additionally, the resident plasmid directs the synthesis of a specific surface protein or lipoprotein that blocks The entry of related plasmids into the cell.

Plasmids confer a wide array of specialized traits upon their host cells. Some plasmids function as "resistance factors" (R-plasmids or R-factors), conferring resistance to antibiotics. A classic example is the penicillinase plasmid found in staphylococci, which can be transduced by various Bacteriophages. This plasmid harbors the Gene encoding penicillinase, an enzyme that degrades penicillin and thereby establishes resistance. The transmission and spread of such factors among bacteria (via Horizontal Gene Transfer) pose significant challenges in clinical practice. Other plasmid-encoded genes confer resistance to disinfectants, contribute to pathologies such as staphylococcal impetigo, assist lactic acid bacteria in converting milk into cheese, and enable the degradation of complex compounds such as Hydrocarbons—Applications useful for bioremediation of oil spills or the PRODUCTION OF SINGLE-cell protein from petroleum.

1.7 Individual Growth and Asexual Cell Reproduction

The surface-area-to-volume ratio of bacterial cells is exceptionally high, which facilitates the rapid uptake of nutrients from the environment via diffusion and active transport. Under favorable conditions, bacteria multiply very rapidly. Growth depends primarily on temperature and pH, nutrient availability, and Ionic strength. Obligate aerobes strictly require oxygen, whereas obligate anaerobes cannot tolerate its presence.

Upon reaching a critical size, bacteria undergo asexual reproduction (binary fission), dividing to produce two daughter cells. The trigger for division is dictated by The ratio of nuclear volume to cytoplasmic volume. Prior to division, DNA replication takes place, during which mesosomes anchor The Genome in a specific position (see Cell Structure diagram). Mesosomes may also attach to nascent septa between daughter cells and play a role in synthesizing cell wall components. In the fastest-growing bacteria, division occurs every 20 minutes; this interval between divisions is termed the generation time.

Septum formation (Fig. 14) initiates after the completion of DNA replication, followed by a 20-minute interval (known as the D period) leading up to cell division. The standard duration required for complete chromosomal replication is approximately 40 minutes, regardless of growth rate. These parameters dictate the timing of DNA replication initiation across various growth rates. For example, if the cell doubling time is 30 minutes, DNA replication concludes 20 minutes before division, and initiation occurs 40 minutes prior to that. Consequently, the initiation of Chromosome replication destined for the next generation coincides with the division event that gave rise to their progenitors. Evidently, initiations must occur in such cultures every 30 minutes.

Fig. 14. Schematic representation of the bacterial Cell Cycle, illustrating the temporal coordination of DNA replication initiation, septum formation, and cell division within a 50-minute cycle.

If the generation time is changed to 50 min. (Fig. 14), DNA replication for the daughter cells is initiated 60 min. prior to their Separation, i.e., 10 min. before the division that yields the parent cell. Thus, we can see that DNA replication can be initiated at any point in the cell cycle, but this point remains fixed for any given generation time. Throughout its lifespan, a cell undergoes various states: a growth phase, as well as phases of preparation for division and division itself. The cell cycle—the transition from cell division to the synthesis of cellular components, and then back to division—can be represented schematically as a cycle comprising several distinct phases (Fig. 15).

Fig. 15. The prokaryotic cell cycle.

Following division, the cell enters a phase of Protein Synthesis AND growth, known as the G₁ phase.

To enter mitosis, a cell must reach a critical mass: the size of newly formed daughter cells initially varies much more widely than at a later stage, when these cells re-enter mitosis. This occurs because smaller cells remain in the G₁ phase until they reach approximately the same size as larger cells. Evidently, a regulatory mechanism governs the mass of cells entering mitosis, even though it operates with a considerable margin of tolerance.

Some cells from this phase transition into the G₀ phase; these cells remain functional and eventually die without dividing (e.g., erythrocytes). However, the majority of cells, having accumulated the necessary substances, restored their size—and sometimes without any size change from the preceding division—begin preparing for the next division. This phase is designated as the S phase (the DNA synthesis phase); subsequently, once the chromosomes have duplicated, the cell enters the G₂ phase, which is the preparation phase for mitosis. Mitosis (cell division) then takes place, and the cycle repeats anew. The G₁, G₂, and S phases collectively constitute interphase (i.e., the phase between cell divisions).

Cellular life and the transition from one phase of the cell cycle to another are regulated by fluctuations in the concentrations of cyclin proteins, as illustrated in Figure 16.

Fig. 16. The Cell life cycle is regulated by cyclin proteins, the concentrations of which vary across different Phases of the cycle. The thickness of the colored sectors corresponds to the cyclin concentration.

1.8 Sexual Reproduction or Genetic Recombination

Sexual reproduction also occurs in bacteria, albeit in its most primitive form. Bacterial sexual reproduction differs from that of eukaryotes in that bacteria do not form Gametes, nor do they undergo cellular fusion. However, the quintessential event of sexual reproduction—namely, the EXCHANGE OF GENETIC material—does take place. This process is termed genetic recombination. A segment of DNA (very rarely the entire DNA) from a donor cell is transferred into a recipient cell, whose DNA is genetically distinct from that of the donor. In the process, the transferred DNA replaces a portion of the recipient's DNA. Enzymes that cleave and rejoin DNA strands participate in this DNA replacement mechanism. Consequently, a recombinant DNA molecule is formed, containing genes from both parental cells. Such DNA is referred to as recombinant. The offspring, or recombinants, exhibit noticeable phenotypic diversity resulting from gene mixing. This diversity is crucial for evolution and represents the primary advantage of sexual reproduction.

Three Methods of obtaining recombinants are known: listed in the order of their discovery, they are transformation, conjugation, and Transduction.

During transformation, the donor and recipient cells do not come into direct contact. This process was discovered in 1928 by Griffith while working with pneumococci—the bacteria responsible for Pneumonia. Pneumococci form two types of colonies that differ in appearance: some are rough (designated as R, from rough), while others are smooth (designated as S, from smooth). R-strains are non-pathogenic and lack capsules; S-strains are pathogenic and possess thick capsules. Griffith observed that if mice were injected with live R-cells and heat-killed S-cells, the mice died within a few days, and live S-cells could be isolated from their Blood. Based on this, Griffith concluded that some factor was released from the dead S-cells, conferring upon the R-cells the ability to form a capsule and protecting them from destruction within the host animal's body. It turned out that this "transformation" is heritable. Since the molecules of "heredity" were still unknown at the time (although proteins were suspected), considerable effort was dedicated to identifying the transforming factor.

In Conclusion, sexual reproduction (in any form) is a relatively rare event in bacteria. However, given the enormous population of bacteria within each colony, sexual reproduction is observed comparatively often. This mode of reproduction is more primitive than in eukaryotes; complete exchange of genomes (total DNA) occurs only during conjugation, which indeed happens only occasionally. Bacterial sexual reproduction is of particular significance because it is precisely through this pathway that resistance to antibiotics and disinfectants is transmitted. The Sexual process in bacteria was described in the mid-twentieth century. This is a process wherein bacteria exchange their Genetic information. Figure 17 presents a diagram of this mechanism, known as conjugation. During conjugation, a cytoplasmic bridge is formed through which a DNA molecule is transferred from one cell to another. Escherichia coli possesses a DNA molecule called the F-factor (fertility factor). The F-factor molecule is capable of integrating into the genomic DNA. The F-factor encodes a specialized protein that forms sex pili. These very pili attach to another cell that lacks the F-factor, which then initiates replication. During replication, two copies of the DNA molecule are produced: one copy remains in the original cell, while the second copy is transferred to the recipient cell. Thus, genetic information is successfully passed from one cell to another.

Fig. 17. The sexual process in bacteria (Beadle and Tatum, 1946; W. Hayes, 1952)

The donor chromosome contains the same genes as the DNA segment transferred into the cell. However, the allelic variants of genes in the original donor cell and the recipient cell may differ. For instance, in the original cell, a gene might encode the Synthesis of the enzyme lactase (which breaks down milk sugar, lactose), whereas in the recipient, the homologous gene is impaired—meaning it fails to encode lactase due to a mutation. As a result, the bacterium is unable to utilize lactose sugar in its environment.

The recipient DNA and the host DNA exchange homologous segments (i.e., segments containing identical genes). A novel combination of genes is thus established within the host cell. A segment carrying a new gene, imported from the donor cell, becomes embedded among its existing genes. This process of DNA segment exchange is known as recombination. Any DNA that fails to integrate into the chromosome during recombination is degraded and lost. The new gene expresses itself, rendering the bacterium capable of cleaving the sugar it could not previously utilize. In such instances, the lactase gene is referred to as a genetic "marker"; it marks a chromosomal locus associated with a specific bacterial trait (the ability to degrade a sugar, which can be detected by researchers). The replication process in E. coli takes 20 minutes, whereas conjugation lasts 3 to 5 minutes. Consequently, only a fragment of the chromosome, rather than the entire genome, manages to transfer within this timeframe. The longer conjugation continues, the larger the transferred chromosomal fragment. This process allows researchers to determine which markers entered the cell if the parental cells initially differed across multiple genes. The F-factor is capable of integrating into various chromosomal sites, and upon the initiation of transfer, different markers are delivered to the recipient cell. Following conjugation, the cells were agitated to disrupt the connecting bridges. This agitation was performed at 2, 3, or 5 minutes, allowing researchers to observe which markers (and consequently, which chromosomal fragments) had transferred by those intervals. Based on these data, a genetic map was constructed illustrating the relative positions of the genetic markers. The Genetic Map of E. coli was established in the 1960s. This map featured marker genes distributed along the circular chromosome, with gene coordinates designated in minutes. The final map compiled in the 1960s spanned coordinates from 0 to 90 minutes. Hence, a prominent microbiologist famously joked that E. coli is an astonishing organism whose lifespan lasts 20 minutes, yet its sexual process takes 90 minutes.

During transduction, a small double-stranded DNA fragment is transferred from a donor cell to a recipient cell via a bacteriophage (a group of viruses). A potential mechanism of transduction is depicted in Figure 18 below.

Certain viruses are capable of integrating their DNA into bacterial DNA; such integrated DNA replicates synchronously with the host DNA and is passed from one bacterial generation to the next. From time to time, this DNA becomes activated and begins encoding The production of new virions. The host (bacterial) DNA is fragmented, and the released pieces are occasionally packaged inside new Viral Particles, sometimes even displacing the virus's own DNA. These novel "viruses," or transducing particles, subsequently transfer DNA into other bacterial cells.

Fig. 18. Mechanism of transduction