Bacterial Cell Wall & Internal Structures

• Bacterial Cell Wall & Structures Internal to the Cell Wall is lecture No. (4) for the Pharm D Program (2024/2025).
• The aim is to provide students with knowledge about the functions and chemical composition of the cell wall and other structures inside bacterial cells.
• Students will discuss the characteristics of bacterial cell walls and other structures, and analyze their importance in the survival of both pathogenic and non-pathogenic bacteria.
• Topics include Cell Wall, Cytoplasmic membrane, Cytoplasm, Nuclear area (Nucleoid), Ribosomes, and Bacterial spores.

Functions of Cell Wall

• A complex semi-rigid structure responsible for cell shape
• The cell wall surrounds the plasma membrane, protecting the interior of the cell from environmental changes, especially hypotonic solutions.

Additional Importance of Cell Wall

• Contributes to the ability of some species to cause diseases (pathogenicity)
• The chemical composition differentiates major types of bacteria
• Composed of unique components found nowhere else in nature
• Constitutes one of the most important sites for attack by antibiotics
• Provides ligand and receptor sites for drugs or viruses

Basic Structure of Cell Wall

• A macromolecular network of a mucopeptide called peptidoglycan or murein
• Peptidoglycan is composed of horizontal layers of an alternating polymer of N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM), cross-linked by glycosidic bonds.
• NAG & NAM are synthesized in the cell cytoplasm and transported through the cytoplasmic membrane by lipid carriers.
• Horizontal layers of peptides are linked by vertical layers of peptides, where NAM is attached to a tetrapeptide of both L- and D- amino acids
• Two of these amino acids are unique to bacteria like Diaminopimelic acid
• Gram-positive bacteria contain many peptidoglycan layers that form a thick rigid structure, in contrast to the thin peptidoglycan layer in Gram-negative cell walls

Cell Wall Synthesis

• Disaccharide pentapeptides are synthesized in the cytoplasm and transported across the cytoplasmic membrane on a lipid carrier
• Isomerase converts two molecules of L-alanine to D-alanine, and a ligase joins them together
• Insertion of the disaccharide pentapeptide into the cell wall at a growing point is done by a transglycosylase
• Fusion of these precursors through their peptides is done by a transpeptidase
• Glycan strands are cross-linked via their peptide chains to the mature peptidoglycan wall and transpeptidases are referred to as penicillin-binding proteins.

Special Structures in Gram-Positive Bacteria

• Teichoic acid is sorbitol or glycerol linked by phospho-diester bonds.
• Due to their negative charge, teichoic acids regulate the movement of cations into and out of the cell, and they also have a role in cell growth by preventing extensive wall breakdown.
• Teichoic acid serves as a surface antigen and virulence factor for pathogenic Gram-positive bacteria
• This provides much of the cell wall's antigenic specificity, which helps in bacterial identification.

Special Structures in Gram-Negative Bacteria

• Contains an outer membrane surrounding the peptidoglycan layer and composed of 3 layers (lipoprotein – phospholipid bilayer – lipopolysaccharide: LPS)
• LPS is formed of an outer polysaccharide somatic e.g. "O" antigen that could be used to identify certain m.o.
• Phospholipid called lipid A, responsible for the endotoxic (pyrogenic) effects causing fever and shock.
• Functions by creating a barrier against antibiotics (e.g. penicillin), digestive enzymes (e.g. lysozyme), detergents, heavy metals, bile salts, and certain dyes.
• Nutrients pass through to sustain metabolism of the cells
• Permeability of the outer membrane is due to channel proteins (porins)

Functions of Porins

• Allow passage of hydrophilic molecules (nutrients) up to a molecular weight of 750 daltons such as nucleotides, disaccharides, peptides, amino acids, vitamins and iron
• Exclude passage and entry of larger molecules or harmful hydrophobic compounds (such as bile salts in the intestinal tract).

Periplasmic Space

• The space between outer membrane & cytoplasmic (plasmic) membrane.
• It contains a single peptidoglycan layer, degradative enzymes (e.g., β-lactamase to destroy antibiotics), & transport proteins.

Gram Stain Procedure

• Heat/Dry
• Crystal violet stain
• Iodine Fix
• Alcohol decolorization
• Safranin stain

Cell Wall Damage

• Peptidoglycan is only found in bacteria so its synthesis is a good drug target
• Lysozyme found in several body secretions degrades peptidoglycan
• Inhibition of mucopeptide (peptidoglycan) biosynthesis often produces wall-less bacteria and in Gram positive cells, this forms a protoplast via complete cell wall removal
• In Gram negative cells, this forms a spheroplast, maintaining some cell wall
• Both spheroplasts and protoplasts are cocci in shape and rupture easily in hypotonic solutions
• When penicillin or lysozyme is removed, spheroplast forms again a cell wall
• Certain Gram-negative bacteria form stable spheroplasts (L-form), capable of growing and dividing

Main Differences Between Gram-Positive and Gram-Negative Cell Walls

• Gram-positive bacteria stain violet and have a thick cell wall (20-80 nm)
• They lack a periplasmic space and outer membrane, and have 1 layer (murine)
• They have >50% peptidoglycan, high degree of cross linking, teichoic acid, 0-3% lipid/lipoprotein, and no lipopolysaccharide
• More sensitive to lysoszyme and penicillin
• Gram-negative bacteria stain red and have a thin cell wall (10 nm)
• They have a periplasmic space and outermembrane, and 2 layers (murine + outer membrane)
• They have 10-20% peptidoglycan, low degree of cross linking, no teichoic acid, 58% lipid/lipoprotein, and lipopolysaccharide
• Less sensitive to lysoszyme and penicillin

Atypical Cell Walls

• Mycobacteria consists of acid-fast bacteria who's cell walls such as Mycobacterium tuberculosis (T.B.), consist of as much as 60% mycolic acid, a waxy lipid, whereas the rest is peptidoglycan.
• Other bacteria with atypical cell walls include protoplasts, spheroplasts, and L-form bacteria.

Structures Internal to the Cell Wall

• Cytoplasmic membrane (Cell membrane, Plasma membrane)

Structure of Cytoplasmic (Plasma) Membrane

• A thin structure lying inside the cell wall, enclosing the cytoplasm of the cell
• Prokaryotes consist primarily of phospholipids (arranged in two parallel rows) called a phospholipid bilayer and proteins
• Each phospholipid molecule contains a polar head composed of a hydrophilic phosphate group and glycerol, and non-polar hydrophobic tails (fatty acids)
• The polar heads are on the surfaces, and the non-polar tails are in the interior of the bilayer.
• Prokaryotic plasma membranes are less rigid than eukaryotic membranes because they lack sterols, except for wall-less prokaryote Mycoplasma, which contains membrane sterols.
• Protein molecules (60-70% of the membrane) can be arranged in peripheral proteins (inner/outer surface) or integral proteins (penetrate membrane).

Function of Membrane Proteins

• Peripheral proteins act as enzymes/support/mediators of changes in membrane shape during movement.
• Integral proteins often function as channels for substances to enter/exit the cell.
• The phospholipid and protein molecules freely move within the membrane (dynamic arrangement) known as the fluid mosaic model.

Functions of Cell Membrane

• Selective permeability: serves as a selective barrier through which materials enter and exit the cell.
• Large molecules (such as proteins) cannot pass, but smaller molecules (such as water, oxygen, carbon dioxide, and some simple sugars) usually pass easily.
• Substances that dissolve easily in lipids (O2, CO2, and non-polar organic molecules) enter/exit more easily because the membrane consists of phospholipids.
• Permeability is either through passive or active processes
• Production of energy (ATP) and cell wall synthesis

Movement of Materials Across Membranes

• Large molecules broken down by extracellular enzymes into simpler subunits
• These are transported by permeases enzymes into the cell

Passive Processes

• Substances cross with the concentration gradient, without any energy (ATP) expenditure
• Includes simple diffusion, facilitated diffusion, and osmosis.
• Simple Diffusion: Movement of molecules/ions from high to low concentration until equilibrium; it is directly proportional to the concentration gradient and hydrophobicity
• Facilitated Diffusion: Substance combines with a carrier protein in the plasma membrane (permeases); no energy needed
• Osmosis: Net movement of solvent molecules across a selectively permeable membrane from a high concentration of solvent molecules to a low concentration to restore equilibrium in osmotic pressure, and osmotic pressure is the pressure required to prevent the water movement.

Osmotic Solutions

• Isotonic: Water moves in and out of the cell at the same rate with no net change
• Hypotonic: Bacteria live in hypotonic solutions, swelling occurs but is contained by cell wall. Cells with weak walls may burst or undergo osmotic lysis.
• Hypertonic: Bacteria shrink and collapse because water leaves (plasmolysis).

Active Processes

• Use energy (ATP) to move substances against the concentration gradient via active transport or group translocation
• Active Transport: Uses energy (ATP) & carrier proteins in the plasma membrane
• Group Translocation: Chemically altered substance across the membrane

Cytoplasm

• The substance inside the plasma membrane (80% water) includes soluble proteins water, enzymes, nucleic acids & nucleotides, CHO, lipids, inorganic ions and other compounds.
• The major structures are DNA, ribosomes, and inclusion bodies.

Nuclear Area

• Consists of a prokaryotic chromosome is one long, closed, circular molecule of double-stranded DNA, free in the cytoplasm without a nuclear membrane.
• The chromosome contains cell structure/function information, with the number of genes reaching up to several thousand
• Plasmids are small, non-essential, extra-chromosomal DNA molecules that replicate independently
• Plasmids equal approximately 1/10 the size of the bacterial chromosome & carrying 5-100 genes, can code for virulence/antibiotic resistance
• Can be transferred between cells during bacterial recombination, forming important tools in industrial microbiology/biotechnology via genetic engineering
• The total DNA content of a prokaryote is referred to as the cell genome

Ribosomes

• The sites of protein synthesis
• Composed of 2 subunits, each consisting of protein & ribosomal RNA (rRNA)
• Prokaryotic differ from eukaryotic ribosomes in the number of proteins and rRNA molecules
• Prokaryotic ribosomes are called 70S (30S and 50S subunits), eukaryotic cells are 80S ribosomes (60S & 40S subunits).
• The 70S ribosome small subunit (30S) contains one molecule of rRNA & > 20 ribosomal proteins; larger subunit (50S) contains two molecules of rRNA & > 30 ribosomal proteins.
• The S refers to Svedberg units, expressing the relative rate of sedimentation via centrifugation

Endospores

• Formed only when environmental conditions are unfavorable
• Protect the bacterial cell against unfavorable conditions.
• Resist drying, heat, freezing, radiation (UV, X-Ray) & toxic chemicals.
• Spores can resist boiling for 2 hours, whereas vegetative cells die at 80°C.
• Sporulation/sporogenesis: endospore formation within a vegetative (parent) cell takes several hours
• Germination: spores into vegetative cells via protective layer break down, water entrance, and favorable conditions placement (nutrient media).
• Contains calcium, spore core is highly dehydrated, and contains DNA, RNA, enzymes, and a few other small molecules.
• The shape, position & size varies (spherical, oval/elliptical) within a central, subterminal, terminal, bulging or non-bulging range
• Resistance of spores is due to the impermeability of the spore wall, high calcium and dipicolinic acid content, low water content, and low dormancy.