MICROBIOLOGY Study Guide - 2012

CHAPTER 3. BACTERIAL MORPHOLOGY

3.3. PROKARYOTIC CELL STRUCTURE

The Fine Structure of The Cell is studied using an Electron microscope and a microtome, which allows for the preparation of ultrathin cell sections. The core structures of a bacterial cell include The Cell wall, cytoplasmic membrane, Cytoplasm with inclusions, and nucleoid. Some Bacteria also possess additional structures: a capsule, flagella, and pili. Certain species of bacteria are capable of forming endospores (Fig. 3).

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Fig. 3. Diagram of BACTERIAL Cell Structure:

1 — cell wall; 2 — cytoplasm with Ribosomes; 3 — CPM; 4 — nucleoid; 5 — polyphosphates; 6 — Lipids; 7 — mesosomes; 8 — capsule; 9 — sulfur inclusions; 10 — flagella

The cell wall is a thin, colorless structure that gives the cell its defined shape and, together with the adjacent cytoplasmic membrane, enables it to withstand high intracellular pressure. The Introduction/4.html">Prokaryotic Cell wall performs diverse functions: it maintains a constant cell shape, participates in Cell Division, mechanically protects the cell from environmental stressors, and prevents the excessive influx of Water. The cell wall accounts for 5 to 20% of the cell's dry weight.

The primary framework of the Bacterial cell wall is a polymer known as peptidoglycan or murein (from Latin murus — wall). The ability to synthesize this type of polymer is unique to prokaryotes (Fig. 4).

Fig. 4. Diagram of a murein fragment structure

Murein is a heteropolymer consisting of parallel glycan molecules cross-linked by peptide bonds. The glycan is formed by alternating residues of N-acetylglucosamine and N-acetylmuramic acid linked by р (1 → 4) glycosidic bonds. The muramic acid residues are attached to Amino Acids via lactyl groups (Fig. 5).

Fig. 5. Single-layer structure of peptidoglycan (Krasilnikov, 1974)

The peptide component of murein typically includes L-Alanine, D-glutamic acid, D-alanine, and a diamino acid specific to prokaryotes: meso-diaminopimelic acid. In some bacteria, this may be replaced by either L-Lysine or L-Ornithine. Diamino acids play a crucial role in the intermolecular cross-linking of glycan layers. The heteropolymer chains are linked together into a murein sacculus via peptide bridges. Teichoic Acids, which are polymers of ribitol phosphate or glycerol phosphate, are covalently bound to murein. Teichoic acids are located on both sides of the peptidoglycan layer, with a lower concentration on the outer side compared to the side adjacent to the cytoplasmic membrane.

It is worth noting that the bacterial cell wall contains substances absent in animals and plants—namely murein, teichoic acids, and meso-diaminopimelic acid (which is never found in Proteins).

The structure of the cell wall was largely elucidated through The Study of the effects of Lysozyme and penicillin on bacteria. Discovered by

A. Fleming, lysozyme is a bactericidal enzyme found in tears, nasal mucus, egg white, and human breast milk. Lysozyme has also been isolated from bacteria and Bacteriophages. When lysozyme is applied to a suspension of Gram-positive bacteria, rapid clearing of the suspension is observed. Lysozyme cleaves the glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid in murein, thus functioning as a muramidase enzyme. Alongside lysozyme, many Other Enzymes can lyse the murein framework.

The antibiotic penicillin acts primarily on Gram-positive bacteria. It interferes with the cross-linking of murein by cleaving the peptide bond. However, the bactericidal effect of penicillin is observed only in actively dividing Cells; older cells remain viable. When a bacterial cell is exposed to penicillin, specialized morphological forms emerge: protoplasts (where the cell wall is completely lysed) and spheroplasts (where the cell wall is partially destroyed). Protoplasts and spheroplasts retain their METABOLISM, growth capacity, and sometimes the ability to divide. Such bacteria are commonly referred to as L-forms. Morphologically, they appear as spherical or irregularly shaped cells ranging from ultrafine to giant sizes.

Gram staining of bacteria. Based on their Chemical Composition and fine cell wall structure, prokaryotes are divided into two major groups: Gram-positive and Gram-negative. In 1884, the Danish scientist Hans Christian Gram proposed a tissue staining method that later became a cornerstone in practical microbiology as one of the most important morphological criteria for bacterial species identification, known as Gram staining. The Essence of the technique is as follows: cells are heat-fixed onto a Glass slide, the smear is stained with a basic violet dye (crystal violet or gentian violet), the dye is rinsed off, and the slide is treated first with an iodine solution and then with an organic solvent (alcohol or acetone), followed by counterstaining with a contrasting dye such as fuchsin.

Iodine forms a stable colored complex with the violet dye. During alcohol Treatment, Gram-positive bacteria retain this complex within the cell, remaining purple, whereas in Gram-negative bacteria, the colored complex is washed out, decolorizing the cell. Subsequent counterstaining with fuchsin turns the Gram-negative bacteria red.

The cell wall of Gram-positive bacteria is quite robust, measuring 20–80 nm in thickness. It has a homogeneous, tubular structure penetrated by pores and adheres tightly to the cytoplasmic membrane. Murein accounts for 30–70% of the dry weight of the cell wall. Teichoic acids are bound to murein; they are found exclusively in Gram-positive bacteria and consist of ribitol- or glycerol-based polymers whose residues are linked by phosphodiester bonds. Free hydroxyl groups in the alcohol molecules may be substituted with residues of D-alanine, glucose, N-acetylglucosamine, and Other Compounds.

Alcohol treatment during Gram staining causes murein to swell and the pore diameter of the cell wall to decrease, generally reducing its permeability and preventing the colored complex from washing out of the cell.

The cell wall of Gram-negative bacteria is multilayered and more chemically diverse (Fig. 6). The inner layer of the cell wall consists of murein, which accounts for 1 to 10% of its dry weight, with a thickness of only 2–3 nm. The murein layer is separated from the membrane by a periplasmic space containing various enzymes. Lipoproteins are covalently linked to the murein layers, with their lipophilic ends facing outward. The outer layer of the cell wall (outer membrane), which is 8–10 nm thick, is composed of Phospholipids, lipopolysaccharides, and proteins, and is linked to murein via lipoproteins. The hydrophobic ends of the phospholipids and lipopolysaccharides face inward, while their hydrophilic heads face outward. Embedded within The Lipid Bilayer of the outer membrane are porin proteins that span the entire layer. Porins allow hydrophilic low-molecular-weight substances (up to approximately 6,000 Da) to pass through the membrane. The lipopolysaccharide (LPS) of the outer membrane consists of three components: lipid A, a core region (or core oligosaccharide), and an O-specific polysaccharide side chain known as the O-antigen. Lipid A confers toxicity to the lipopolysaccharide, making LPS one of the most potent bacterial endotoxins.

Fig. 6. Model of the cell wall structure in Gram-negative bacteria (Schlegel, 1987):

CM — cytoplasmic membrane; PP — periplasmic space; M — murein; OM — outer membrane

Lipopolysaccharides play a crucial role in bacteriological Diagnostics and epidemic tracking. The O-specific side chains of LPS from different bacterial species and strains vary in composition. They determine the serovar (a bacterial variety identified serologically using immune serum) of a specific bacterial strain.

Because Gram-negative bacteria have a thin murein layer, it does not play a significant role in maintaining wall permeability. Furthermore, the high lipid content in their cell wall promotes the washing out of the stained complex from the cell when the smear is treated with alcohol during Gram staining.

Table 3 lists several representatives of Gram-positive and Gram-negative bacteria.

Table 3. Representatives of Gram-positive and Gram-negative bacteria

It should be emphasized that Gram staining is not always a completely reliable method for differentiating these two groups of bacteria. Some Gram-positive bacteria rapidly lose their ability to retain the gentian violet-iodine complex as they age. Therefore, young cultures (18–24 hours old) must be used for Gram staining. In addition, certain Gram-positive bacteria may yield a gram-variable reaction.

Prokaryotes lacking a cell wall do occur in nature. These are the smallest prokaryotes—Saprophytes or parasites capable of independent Replication within a host Organism and responsible for chronic infections. They are called Mycoplasmas. Specifically, Mycoplasma pneumoniae causes acute respiratory-like infections in humans. Due to the absence of a cell wall, these bacteria lack a fixed shape and are highly pleomorphic.

Prokaryotes have been discovered whose cell wall Composition and Structure differ significantly from the peptidoglycan described above. These microorganisms belong to the group of archaea. For instance, in halophilic archaea of the genus Halobacterium, the cell wall consists mainly of Glycoproteins, whereas in archaea of the genus Halococcus, it is made of Heteropolysaccharides. Methanogenic bacteria possess Three types of cell walls: those composed of pseudomurein, those built from protein globules, and those made of heteropolysaccharides.

The unique Chemical composition of the prokaryotic cell wall has enabled The Development of numerous medications that specifically target this bacterial structure. Such drugs include penicillin, bacitracin, novobiocin, and Other Antibiotics.

The cytoplasmic membrane (CM) (from Latin membrana — Skin, parchment) is located between the cell wall and the cytoplasm. It is an essential structural element of any cell and represents a protein-lipid complex containing 50 to 75% proteins and 15 to 45% lipids. The thickness of the CM is typically 4–7 nm.

The membrane contains three classes of lipids: phospholipids, Glycolipids, and Steroids (Cholesterol). Phospholipids play the primary structural role in the CM. They form a bilayer structure in which the hydrophilic phospholipid heads face outward, while the hydrophobic "tails" (typically two fatty acid chains) face inward. Protein globules float within the phospholipid layer, being either fully embedded in it (integral proteins) or located On the surface (peripheral proteins) (Fig. 7).

Fig. 7. Model of The Plasma Membrane structure:

1 — carbohydrate fragments of glycoproteins; 2 — phospholipid bilayer; 3 — integral protein; 4 — phospholipid heads; 5 — peripheral protein; 6 — cholesterol; 7 — phospholipid fatty acid tails

Protein globules form channels that function as pumps, utilizing ATP to drive the movement of substances and ions (sodium, potassium, calcium, etc.). Sodium-potassium pumps transport Na⁺ out of the cell and K⁺ into the cell (antiport) against their chemical gradients. Ion transport is carried out by the primary component of the pump—sodium-potassium-dependent ATPase via ATP Hydrolysis. For every hydrolyzed ATP molecule, three sodium ions and two potassium ions are transported.

The cytoplasmic membrane is a dynamic structure with mobile components; hence, it is regarded as a fluid structure. The membrane plays a vital role in cellular metabolism. It serves as the cell's osmotic barrier, controlling both the uptake of nutrients into the cell and the efflux of metabolites. The membrane is also responsible for bioenergetic processes, as Electron Transport and Oxidative Phosphorylation enzymes (Cytochromes, iron-sulfur proteins, and other components) are localized within or on The surface of the membrane.

The CM participates in cellular energy conversions. Bacteria derive energy through Respiration or Photosynthesis, while the CM houses the Electron Transport Chain carriers that generate electrochemical energy (Δp_H⁺), which is utilized by the cell for various purposes, including the synthesis of chemical energy—ATP. The membrane also contains enzyme complexes that catalyze The conversion of electrochemical energy into ATP (Δp_H⁺ → ATP).

Furthermore, the replication Origin of the bacterial chromosome is localized at the cytoplasmic membrane.

Mesosomes. The growth rate of the cytoplasmic membrane typically outpaces that of the cell wall, resulting in the membrane frequently forming numerous inward invaginations of various shapes known as mesosomes. Bacterial mesosomes vary considerably in shape, size, and intracellular Location. The simplest mesosomes are vesicular (bubble-like), whereas more complex ones include lamellar (platelike) and tubular mesosomes. Based on their cellular position, a distinction is made between mesosomes formed in the zone of cell division and transverse septum formation, mesosomes to which the nucleoid attaches prior to cell division (nuclear mesosomes), and mesosomes formed by the invagination of peripheral Regions of the CM (peripheral mesosomes).

The cytoplasm is separated from the cell wall by the cytoplasmic membrane and constitutes a complex colloidal system consisting of 80–85% water, along with soluble proteins, enzymes, RNA, substrates, and metabolic reaction products. The cytoplasm contains ribosomes and reserve nutrients such as Polysaccharides, poly-β-hydroxybutyric acid, polyphosphates, sulfur, iron, and other compounds.

Ribosomes are ribonucleoprotein particles with a diameter of 15–20 nm, composed of 2/3 RNA and 1/3 proteins. Their primary function is Protein Biosynthesis. The number of ribosomes in a cell ranges from 5,000 to 90,000. Prokaryotic ribosomes have a sedimentation coefficient of 70 Svedberg units (S) and are referred to as 70S ribosomes. Each ribosome consists of two subunits: 30S and 50S. During active cellular Protein Synthesis, regular chains of ribosomes resembling beads are formed; these are called polyribosomes or Polysomes.

The nucleoid of prokaryotes is represented by a DNA molecule that forms a closed-loop structure, compactly packed and occupying a specific region in the cytoplasm. The Molecular Weight of the nucleoid is (1–3) × 10⁹ Da. The nucleoid is not separated from the cytoplasm by a nuclear membrane. The DNA molecule concentrates almost all the Genetic information of the cell, which is why it is also termed the bacterial chromosome. Typically, a bacterial cell contains a single chromosome. Bacteria with large cells (such as filamentous cyanobacteria) often contain multiple nucleoids—up to 8.

Extrachromosomal genetic elements. Many bacteria harbor extrachromosomal genetic elements: Plasmids, temperate phages, Transposons, and IS elements.

Plasmids are small, non-chromosomal DNA molecules responsible for specific traits. For instance, the fertility factor—the F-factor (from fertility)—is a plasmid that carries the genes controlling conjugation. The R-factor, or resistance factor (from resistance), carries genes responsible for bacterial drug resistance. Plasmids can also determine bacterial virulence, as seen in the causative agents of plague, tetanus, gas gangrene, and others.

Many bacteria synthesize proteins that inhibit the growth of related species or competitors. These specific-action proteins are called bacteriocins. Their synthesis is encoded by specialized plasmids or bacteriocinogenic factors. Bacteriocins have been isolated from cells of Escherichia coli (colicins), Pseudomonas aeruginosa (pyocins), Bacillusmegatherium (megacins), and Lactobacillus acidophilus (lactocins).

Plasmids apparently also control the Synthesis of specific proteins known as Prions—novel agents of infectious diseases. They were discovered in the early 1970s by Stanley Prusiner, a neurobiologist at the University of California, San Francisco. According to Prusiner's hypothesis, environmental factors cause the protein to undergo a genetic mutation, altering its stereostructure. It acquires infectious properties, leading to neuronal death and The formation of microscopic vacuoles, resulting in a spongy appearance. This disrupts The Nervous system, hence the name of the condition: spongiform encephalopathy or spongy encephalitis. For his groundbreaking research on the pathogenic agent causing transmissible spongiform encephalopathy, or "mad cow disease" in cattle, Prusiner was awarded the Nobel Prize in Physiology or Medicine in 1997.

Transposons and IS elements, also known as Mobile Genetic Elements, are linear double-stranded DNA molecules ranging from 200 to 6,000 Base Pairs in length. These elements can integrate into various sites of the bacterial chromosome or migrate between the chromosome and a plasmid. Characteristically, transposons and IS elements are incapable of autonomous replication; they replicate synchronously with the host chromosome or plasmid. The transfer of mobile elements (transposition) occurs at a frequency of 10^-4 to 10^-6. IS elements do not encode any phenotypic traits; they contain only the information necessary for their intracellular transposition. More complex transposons carry genes responsible for resistance to antibiotics, heavy Metal Ions, and other inhibitors.

Temperate phages and certain plasmids act as vectors for transferring mobile elements between cells. The integration of mobile elements into the bacterial chromosome gives rise to mutant cells with altered nucleotide triplets, which consequently disrupts the Transcription process.

Reserve substances. In prokaryotes, reserve Materials include polysaccharides, lipids, polyphosphates, sulfur compounds, and hydrocarbon granules. These substances accumulate within the cell when the growth medium contains the corresponding compounds. During periods of nutrient and energy depletion, they enter metabolic pathways, thereby extending cell survival.

Among polysaccharides, Glycogen and starch are found in bacterial cells. Anaerobic spore-forming bacteria, such as clostridia, are capable of accumulating a starch-like substance known as granulose. Unlike cell wall polysaccharides, all Reserve Polysaccharides are synthesized from α-D-glucose.

Lipids accumulate as granules and fat droplets that exhibit strong light refraction under a Light Microscope. The lipids of many bacteria consist of poly-β-hydroxybutyric acid. Poly-β-hydroxybutyric acid is synthesized by numerous aerobic bacteria, cyanobacteria, and facultative phototrophic bacteria. Mycobacteria accumulate Waxes (esters of Higher Fatty acids and alcohols) as reserve lipids.

Polyphosphates. Many bacteria are capable of accumulating phosphoric acid in the form of polyphosphate granules. Such granules were first described in Spirillum volutans, which is why they are also called volutin granules or metachromatic granules; when bacterial smears are stained with methylene blue, the cytoplasm stains blue while the polyphosphate granules stain reddish-purple. Cells utilize polyphosphates as a source of phosphorus and energy.

Sulfur is accumulated by sulfur bacteria in the presence of hydrogen sulfide in the medium and is oxidized to sulfate once the hydrogen sulfide is depleted. For aerobic colorless sulfur bacteria, sulfur serves as an energy source, whereas for anaerobic phototrophic purple sulfur bacteria, it acts as an electron donor.

Flagella. The cell surface of many bacteria bears structures that ensure their motility in a liquid environment. The presence, number, dimensions, and arrangement of flagella are characteristic traits of a given bacterial species and hold significant taxonomic value. However, these features are variable and depend on the age of the culture and cultivation conditions. Older cells are typically non-motile, so young cultures are used to determine motility. In rod-shaped bacteria, flagella may be located polarly (at the cell poles) or laterally (along the cell's lateral surface). Depending on the number of flagella and their surface arrangement, they are classified into the following types:

✵ monotrichous (polar monotrichous) — a single flagellum is attached to one pole of the cell (Fig. 8, 1);

✵ lophotrichous (polar polytrichous) — a tuft of flagella is attached to one pole of the cell (Fig. 8, 2 — a, b, c);

✵ bipolar monotrichous — a single flagellum is located at each pole of the cell;

✵ amphitrichous (bipolar polytrichous) — a tuft of flagella is present at each pole of the cell (Fig. 8, 3);

✵ peritrichous — numerous flagella are distributed across the entire surface of the cell (Fig. 8, 4).

Fig. 8. MAIN TYPES OF flagellation in bacteria (Schlegel, 1987)

In spiral-shaped forms, flagella were discovered later using Electron Microscopy. This indicates that spiral forms can move not only through body flexion but also with the aid of flagella. Spherical bacteria, with the exception of Sarcina ureae, lack flagella.

Flagella are helically coiled filaments composed of the specialized protein flagellin. They are 12–20 nm in thickness and range from 3 to 20 µm in length. A flagellum consists of three parts: a helical filament (fibril), a hook, and a basal body. The filament is attached to the hook, which is a curved protein cylinder. The hook, in turn, connects to the basal body embedded in the plasma membrane.

Flagella perform a rotational movement similar to a ship's propeller at a speed of 10–20 s^-1. When the cell moves forward in a straight line, the flagellum rotates counterclockwise, whereas backward movement involves clockwise rotation. If a cell bears multiple flagella, they coalesce into a bundle during movement.

It has been established that motile bacteria can respond to subtle environmental changes by migrating in a specific direction. Such directional movements are termed taxis, which includes chemotaxis, phototaxis, magnetotaxis, thermotaxis, and viscotaxis. Chemotaxis—movement in a specific direction relative to the source of a chemical substance—attracts the most attention. These chemicals are classified either as attractants (which draw bacteria) or repellents (which drive them away). Attractants may include Vitamins, amino acids, and sugars, whereas repellents include alcohols, alkalis, acids, and phenols. Molecular oxygen acts as an attractant for aerobic bacteria and as a repellent for anaerobes. Nutrient substrates frequently function as attractants.

Pili (fimbriae) are long, non-motile, thread-like structures located on the surface of certain bacteria. Fimbriae are cylindrical, measuring 0.2–2.0 µm in length with an inner diameter of 5–10 nm. They are composed of a protein called pilin. Based on Morphology, antigenic properties, and functions, several types of fimbriae are distinguished. Type 1 fimbriae function in cellular attachment to substrate surfaces or mutual Cell Adhesion. Type 2 (common) fimbriae are responsible for cell Nutrition and Water-Salt Metabolism. The number of common fimbriae can reach several thousand. The term pili is used to designate the third type of fimbriae (F-pili)—sex pili produced by donor cells. Their number is typically 1–3 per cell. F-pili establish contact between donor and recipient cells, forming a conjugation channel through which genetic information is transferred.

Capsule. Many prokaryotes synthesize organic polymers that are deposited on the outer side of the cell wall as an amorphous layer known as a capsule or slime layer. Depending on the thickness of this mucous layer, scientists distinguish microcapsules (visible only under an electron microscope, up to 0.2 µm thick), macrocapsules (slime layers exceeding 0.2 µm in thickness), and slime layers (mucous material surrounding the cell that often surpasses it in thickness). In some saprophytic bacteria, a common capsule encompasses multiple individuals. Aggregations of microorganisms enclosed within a common capsule are referred to as zoogloeae.

The capsule and slime layer perform a protective function, shielding the cell from desiccation and mechanical damage, while also inhibiting host defense mechanisms and bacteriophage penetration.

A capsule or slime layer is not an essential STRUCTURE OF THE bacterial cell. Bacteria continue to multiply even after the mucous layer is removed. Many species exist in both encapsulated and non-encapsulated variants. The presence of a capsule depends on the microbial strain, the age of the culture, and environmental growth conditions. Through mutation, some capsule-forming bacteria can readily lose their capsules and give rise to non-encapsulated forms, a process known as S → R transformation. Colonies consisting of encapsulated cells have a smooth surface and are designated as S-colonies (from smooth), whereas colonies formed by non-encapsulated bacteria have a rough surface and are referred to as R-colonies (from rough).

In most cases, the capsule is composed of homo- or heteropolymeric polysaccharides. The former are built from sugar residues of a single type (glucans, Mannans), while the latter consist of various sugar residues. The capsules of certain Bacillus species are made of Polypeptides, which are polymers of D- and L-glutamic acid. Both the cell wall and the cytoplasmic membrane are involved in the formation of exopolysaccharides of the capsule. However, another mechanism also exists, driven by extracellular enzymes acting on Disaccharides to produce dextrans and levans. Abundant slime production is observed during the growth of Leuconostoc subsp. dextranicum in a sucrose-containing medium. This representative of Heterofermentative lactic acid bacteria rapidly converts the sucrose present in the solution into dextran. This transformation occurs extracellularly and is catalyzed by the extracellular enzyme dextransucrase. Dextran is a polysaccharide composed of α-D-glucose residues linked at the 1,6 position, making it a 1,6-α-glucan.

Streptococci of the species Streptococcus mutans, which are responsible for dental caries, secrete an enzyme that converts sucrose into polyfructose, or levan. This polysaccharide deposits on the tooth surface, trapping acidic Fermentation products generated by the streptococci—predominantly lactic acid.

Currently, extracellular polymers synthesized by specific bacterial species find Practical Applications as Blood Plasma substitutes and for The production of synthetic films.

Capsules are visualized using specialized staining techniques. India ink, Congo red, or nigrosin—compounds that do not penetrate the capsule—are added to the specimen preparation, resulting in negative staining where the light capsule stands out against a dark Background.

Bacterial pigments. Colonies of many bacteria exhibit various colors, such as yellow, orange, pink, red, yellow-green, blue, and others. This coloration results either from the secretion of pigmented products into the external environment or from the pigmentation of the cell itself. The ability to synthesize pigments is genetically encoded and can serve as a diagnostic feature in bacterial identification. These pigments typically belong to different classes of chemical compounds, including carotenoids, pyrroles, azaquinones, and anthocyanins. For instance, the bacterium Serratia marcesscens forms bright red colonies on nutrient media resembling drops of blood, which earned it the historical name "the miracle bacterium" (bacillus prodigiosus). Bacteria of this species synthesize a characteristic red cellular pigment called prodigiosin, which is a tripyrrole derivative. Red pigments can also be synthesized by actinomycetes and purple sulfur bacteria, while pink pigments are produced by micrococci.

Bacteria of the species Pseudomonas aeruginosa secrete pyocyanin, a water-soluble blue phenazine pigment that diffuses into the medium. The bacterium Pseudomonas fluorescens synthesizes a water-soluble green pigment called fluorescein, causing it to form fluorescent yellowish-green colonies when grown on solid media.