GENERAL MICROBIOLOGY - T.P. Pyroh - 2004

5. STRUCTURE OF THE MICROBIAL CELL

5.1. CELL WALLS OF MICROORGANISMS

5.1.2. Structure and Chemical Composition of Prokaryotic Cell Walls

The chemical composition of MICROBIAL Cell WALLS initially attracted the attention of researchers in the field of systematics.

This was based on data concerning qualitative differences in Cell wall composition between eukaryotic and prokaryotic microorganisms, as well as among prokaryotes—between Gram-positive and Gram-negative Bacteria (Table 5.1).

Class="center">Table 6.1

Chemical composition of Microbial Cell Walls

Gram Staining. Differential staining of bacteria with gentian violet was proposed in 1884 by the Danish pharmacologist H.C. Gram. In microbiology, Gram staining is an important taxonomic feature that correlates with other bacterial properties. The Essence of the method is that when bacteria are stained with gentian violet (crystal violet, methyl violet), the dye forms a compound with iodine that is retained within the Cells upon Treatment with alcohol. Such bacteria stain blue-violet and are referred to as Gram-positive.

Bacteria that are decolorized upon alcohol treatment are called Gram-negative. They are subsequently counterstained with a contrasting dye (fuchsin).

In 1978, N.E. Gibbons and R.G.E. Murray proposed classifying true Gram-negative bacteria (eubacteria) into the phylum Gracilicutes, and Gram-positive bacteria into the phylum Firmicutes. However, the terms "Firmicutes" and "Gracilicutes" are not widely used in general microbiology,

Peptidoglycan (glycopeptide, mucopeptide, murein). The primary component of the Introduction/37.html">Bacterial cell wall is peptidoglycan (glycopeptide, mucopeptide, murein). Peptidoglycan is found exclusively in prokaryotes. Exceptions include wall-less eubacteria (Mycoplasmas, L-forms) and archaebacteria—certain methanogens and halophiles (Halobacterium, Halococcus). For halophilic bacteria, the presence of a rigid cell wall is not strictly necessary, as their intracellular content is isosmotic with the external environment.

The specific heteropolymer peptidoglycan consists of: N-acetylglucosamine and N-acetylmuramic acid residues linked together by β-1,4-glycosidic bonds. N-acetylglucosamine is a glucose derivative in which the hydroxyl group at the second carbon atom is replaced by an amino group. N-acetylmuramic acid is an ether of N-acetylglucosamine and D-lactic acid (Fig. 5.2); diamino acids, most commonly meso-diaminopimelic acid, LL-diaminopimelic acid, Lysine, and Ornithine. The presence of such Amino Acids with two amino groups is of fundamental importance for the Spatial Organization of peptidoglycan, as they enable The formation of two peptide bonds between peptide groups within the molecule; Other Amino Acids (D- and L-Alanine, D-glutamic acid, L-Serine, Glycine).

Fig. 5.2. Main Components of the peptidoglycan heteropolymer

A fragment of peptidoglycan is shown in Fig. 5.3. Through peptide bridges, the heteropolymer chains are linked together into a sac-like giant molecule—the murein sacculus (murein net).

The murein sacculus acts as the structural framework of The Cell wall. Gram-positive and Gram-negative bacteria differ in The Structure of this framework, as well as in the content of other cell wall substances.

Fig. 3.3. Structure of peptidoglycan

Cell Wall of Gram-Positive Bacteria. In Gram-positive bacteria, the murein network accounts for 30–70% of the dry weight of the cell wall (reaching up to 40 layers in thickness). Instead of meso-diaminopimelic acid, LL-diaminopimelic acid or lysine is frequently present. In the cell wall of Gram-positive bacteria, Polysaccharides, when present, are covalently linked. The lipid and protein content is low. The cell wall Proteins of Gram-positive bacteria contain a narrower range of amino acids (4–12) compared to Gram-negative bacteria (which contain virtually all amino acids found in proteins).

A characteristic feature of Gram-positive bacteria is the presence of Teichoic Acids in the cell wall. Teichoic acids are chains consisting of 8–50 glycerol or ribitol residues linked by phosphate bridges. Within the teichoic acid molecule, the polyol may contain Monosaccharides as substituents. Some teichoic acids contain erythritol or mannitol. It is hypothesized that teichoic acids are linked to murein via phosphate groups in an amide-like fashion. The teichoic acids of certain Gram-positive bacteria contain Fatty acids that form ester bonds with the glycerol residues; these are known as lipoteichoic acids. Teichoic acids are abundant in the cell, in some bacteria accounting for more than half of the cell wall mass.

Functions of teichoic acids:

the phosphate groups of teichoic acids serve as binding sites for magnesium cations, which are essential for numerous enzymatic and physicochemical processes occurring at the cytoplasmic membrane;

teichoic acids are involved in regulating The activity of autolytic Enzymes;

It has been shown that the sugar components of teichoic acids are responsible for binding phages to the cell wall. If a teichoic acid loses its glycosyl substituents for any reason, the bacterial cell becomes phage-resistant;

lipoteichoic acids participate in immunological reactions.

The cell wall of Gram-negative bacteria. In Gram-negative bacteria, the murein layer is single-stranded and accounts for less than 10% of the dry weight of the cell wall. Murein contains only meso-diaminopimelic acid and lacks lysine. Teichoic acids have not been detected in the cell walls of Gram-negative bacteria.

In all Gram-negative bacteria, outside the single- or at most double-layered murein sacculus, there is an additional layer of the cell wall. This is the so-called outer membrane, which consists of proteins, Phospholipids, and lipopolysaccharides (LPS) (Fig. 5.4).

Fig. 5.4. Structural model of the Gram-negative bacterial cell wall

Lipoproteins are apparently covalently linked to murein via diaminopimelic acid. They are oriented with their lipophilic ends outward and are thus anchored in the lipophilic bilayer (through hydrophobic interactions). This same layer contains phospholipids and the hydrophobic ends of lipopolysaccharides. The hydrophilic ends of LPS are oriented outward.

Lipopolysaccharides are complex molecules with a molecular weight exceeding 10,000. They consist of three parts: lipid A, the core (core region), and the O-specific side chain. The LPS of Salmonella typhimurium and other enterobacteria have been studied in considerable detail.

Lipid A consists of a glucosamine disaccharide, the hydroxyl groups of which are ester-linked to fatty acids (C₁₂, C₁₄, C₁₆). This part of the molecule possesses hydrophobic properties. Next is the R-core region—a trisaccharide consisting of three residues of 2-keto-3-deoxyoctonic acid (KDO), which is also linked to phosphoethanolamine. This is followed by two heptose molecules and the outer core region. The latter consists of a branched chain containing glucose, galactose, and N-acetylglucosamine. This basic structure is identical in all salmonellae. The O-specific side chain is attached to the core region. These are long chains consisting of repeating Oligosaccharides that may contain galactose, mannose, rhamnose, abequose, fucose, and other monosaccharides in a sequence that varies from strain to strain.

LPS have gained great importance in bacteriological Diagnostics and epidemic surveillance. It has been found that pathogens of various diseases differ from one another in their O-specific side chains. Minor differences in their composition can be detected using immunological Methods. Based on serological reactions, over a thousand species and strains within the genus Salmonella have been identified. There are so-called local strains of salmonellae that can be identified by their immunochemical characteristics. This often makes it possible to determine where a patient was infected or where an epidemic originated. For example, one can determine whether a patient acquired the infection in the South American or East Asian region.

Functions of the outer membrane. The outer membrane of Gram-negative bacteria performs not only mechanical but also important physiological functions. Embedded in its lipid bilayer—consisting of lipid A, polysaccharides, and phospholipids—are proteins that span the entire layer. These transmembrane proteins are called porins. Porins allow hydrophilic low-molecular-weight substances (with a Molecular Weight of up to about 6,000) to pass through the membrane.

The outer membrane lies adjacent to the murein layer and is linked to it by lipoproteins. Apparently, the murein layer is permeable to various compounds. The space between the murein and The Plasma Membrane is called the periplasmic space. It contains enzymes, including depolimerases (proteinases, Nucleases), peripheral proteins, and so-called binding proteins. The latter participate in The transport of certain substrates into the Cytoplasm and serve as receptors for chemotactic signals. The periplasmic space undoubtedly plays a role in osmoregulation.

Action of Lysozyme and penicillin. The STRUCTURE OF THE cell wall and murein was elucidated in connection with studies on the effects of lysozyme and penicillin on bacteria. Discovered by the English microbiologist A. Fleming in 1922, lysozyme is a bactericidal enzyme found in egg white, nasal mucus, and tear fluid. Lysozyme has also been isolated from bacteria (E. coli, Streptomyces) and Bacteriophages. The action of lysozyme on a suspension of Gram-positive bacteria results in its rapid clearing. Thus, Micrococcus luteus is lysed (dissolved) at a lysozyme concentration of just 1 µg/ml. Lysis of Bacillus megaterium cells requires a concentration of 50 µg/ml, whereas Gram-negative bacteria are dissolved only in the presence of EDTA in the suspension.

Lysozyme cleaves the glycosidic bond in murein between the first carbon atom of N-acetylmuramic acid and the fourth carbon atom of N-acetylglucosamine (see Fig. 5.3). In the process, the polysaccharide chains are broken down into disaccharide fragments. Consequently, lysozyme is an N-acetylmuramidase.

It should be noted that complete destruction of bacterial cells can be prevented by carrying out lysis in an isotonic or mildly hypertonic solution (0.1–0.2 M sucrose). Under these conditions, lysozyme converts the cells into spherical protoplasts that are extremely sensitive to osmotic conditions. Protoplasts are stable in hypertonic and isotonic solutions, but burst in hypotonic ones. Only those spherical cells that lack any cell wall remnants—meaning neither muramic acid nor cell-wall-specific diaminopimelic acid can be detected—should be called protoplasts. Cell wall lysis is not accompanied by Metabolic Disorders.

Besides lysozyme, there are several Other Enzymes that degrade the murein framework, such as muroendopeptidases.

The antibiotic penicillin acts primarily on Gram-positive bacteria (pneumococci and staphylococci) as well as some Gram-negative ones (gonococci, meningococci, enterobacteria), killing them. However, only growing cells are susceptible to its bactericidal action. Resting cells remain alive. The most interesting phenomenon observed under METABOLISM/18.html">The Influence of penicillin is the appearance of so-called forms produced from normal bacterial cells As a result of unbalanced growth in length and width. Consequently, the original rod-shaped cells increase in volume many times over. When growing cells are treated with penicillin in a hypotonic solution, they burst. In iso- and hypertonic solutions, the rods transform into spherical structures called L-forms or spheroplasts. They differ from protoplasts in that they retain cell wall remnants. Penicillin disrupts The process of Cell wall formation.