Microbial Cell Structure and Diversity: Prokaryotic Cells

Comparison of Prokaryotic and Eukaryotic Cells

• Cell Types: All living organisms on Earth produce only two types of cells: Prokaryotes and Eukaryotes.

• Prokaryotes:
      * Etymology: Derived from "pro" meaning primitive, and "karyote" referring to the nucleus (primitive nucleus).
      * Definition: Cells that do not possess a membrane-bound nucleus.
      * Classifications:
          * Eubacteria (true bacteria).
          * Archaebacteria (ancient bacteria).

• Eukaryotes:
      * Etymology: Derived from "eu" meaning true, and "karyote" referring to the nucleus (true nucleus).
      * Definition: Cells that possess a membrane-bound nucleus.
      * Examples: Algae, fungi, protozoa, plants, and animals.

Phylogenetic Classification and the Tree of Life

• Three Domain Classification: Proposed by Carl Woese in the 1970s.

• Basis of Classification: Based on the sequencing of $16s\,rRNA$.

• Domains: The classification system divides life into three distinct domains based on phylogenetic relationships.

Average Chemical Composition of Bacteria

• Water Content: Constitutes $70\%$ of the bacterial cell.

• Dry Weight Content: Constitutes $30\%$ of the bacterial cell.

• Breakdown of Dry Weight Components:
      * DNA: $5\%$ ($MW = 2,000,000,000$).
      * RNA: $12\%$.
      * Protein: $70\%$. These are found in:
          * Ribosomes: Approximately $10,000$ per cell.
          * Protein particles: $MW = 3,000,000$.
          * Enzymes.
          * Surface structures.
      * Polysaccharides: $5\%$.
      * Lipids: $6\%$.
      * Phospholipids: $4\%$.

Prokaryote Cellular Structures Overview

• Appendages:
      * Flagella.
      * Pili.
      * Fimbrae.

• Cell Envelope:
      * Glycocalyx.
      * Cell wall.
      * Cell membrane.

• Cytoplasm:
      * Ribosomes.
      * Inclusion granules.
      * Plasmid.

• Nuclear Material:
      * Nucleoid or chromosome.

The Bacterial Cell Wall: Structure and Function

• Definition: A tough, usually flexible but sometimes rigid layer located outside the cell membrane.

• General Functions:
      * Provides structural support and protection.
      * Acts as a filtering mechanism.
      * Functions as a pressure vessel, preventing over-expansion and lysis when water enters the cell through osmosis.

• Presence in Organisms: Found in plants, bacteria, fungi, algae, and some archaea. Animals and protozoa lack cell walls.

• Chemical Composition of Bacterial Cell Walls:
      * Primarily composed of peptidoglycan (also known as murein).
      * Structure: Polysaccharide chains cross-linked by unusual peptides containing $D$-amino acids.
      * Distinct from plant cell walls (cellulose) and fungal cell walls (chitin).
      * Distinct from Archaea, which do not contain true peptidoglycan.

• Antimicrobial Interactions:
      * Penicillin: Kills bacteria by preventing the cross-linking of peptidoglycan, weakening the wall and causing lysis.
      * Lysozyme: An enzyme capable of damaging bacterial cell walls by physically cleaving the backbone.

Gram-Positive vs. Gram-Negative Cell Walls

• Gram-Positive Cells:
      * Peptidoglycan Layer: Makes up as much as $90\%$ of the cell wall. It is thick, compact, and is the outermost structure.
      * Peptidoglycan Composition: A polymer of alternating $N$-acetylmuramic\,acid\,(NAM) and $N$-acetylglucosamine\,(NAG). Straps of this polymer are linked by tetrapeptides consisting of $L$-alanine, $D$-glutamic\,acid, $L$-lysine, and $D$-alanine attached to NAM.
      * Cross-linking: Highly cross-linked compared to Gram-negative cells.
      * Lipoteichoic acids (LTA): Polysaccharides found only in Gram-positive bacteria. They extend through the peptidoglycan layer to the cell surface and serve as antigenic determinants.

• Gram-Negative Cells:
      * Structure: More chemically complex, thinner, and less compact.
      * Peptidoglycan Layer: Makes up only $5-20\%$ of the cell wall. It is not the outermost layer; it resides in the periplasm between the plasma membrane and an outer membrane.
      * Outer Membrane: Less permeable than the plasma membrane and composed of lipopolysaccharides (LPS).
      * Periplasm: The space between the cell wall and the plasma membrane which controls molecular traffic.
      * Lipopolysaccharides (LPS):
          * Classified as an endotoxin.
          * Components: (1) Lipid A, (2) Core polysaccharide, and (3) $O$ side chain.
          * Antigenicity: The $O$ side chain (O antigen) is used for serological identification, such as serotypes of $E.\,coli$.

Cell Walls of Archaea and Mycobacteria

• Archaea Cell Wall:
      * Lacks peptidoglycan and shows considerable variety.
      * S-layer: The most common type; composed of protein or glycoprotein. It can be $20-40\,nm$ thick (e.g., $Methanococcus$, $Halobacterium$).
      * Polysaccharides: Thick polymers. For example, $Methanobacterium$ contains galactosamine, glucuronic acid, glucose, and acetate. $Halococcus$ contains sulfated glucose and other sugars.
      * Glycoproteins: Negatively charged proteins with many acidic residues (aspartic acid) decorated with various sugars.
      * Pseudopeptidoglycan (Pseudomurein): Found in $Methanobacterium$, $Methanothermus$, and $Methanopyrus$. It consists of alternating repeats of $N$-acetylglucosamine and $N$-acetyltalosaminuronic\,acid with ($\beta\,1-3$) linkages. It is resistant to lysozyme.
      * Specific Structures: $Methanospirillum$ has a protein sheath; $Methanosarcina$ has a chondroitin-like layer called methanochondroitin.

• Mycobacterial Cell Wall (e.g., Mycobacterium tuberculosis):
      * Complex structure including a lipid bilayer, peptidoglycan, and arabinogalactan.
      * Key component: Mycolate / Mycolic Acid.
      * Other components: Porins, acyl lipids, lipomannan (LM), arabinomannan (AM), and lipoarabinomannan (LAM).

Degradation of Peptidoglycan and Cell-Wall Deficient Forms

• Autolysins: Hydrolytic enzymes produced by bacteria themselves to allow for cell enlargement and division. They include glycosidases, amidases, and peptidases.

• Lysozymes: Glycosidases that cleave the bond between $NAM$ and $GlcNAc$ ($N$-acetylglucosamine). Found in egg whites and human secretions like tears. Gram-positive bacteria (GPB) are directly killed; Gram-negative bacteria (GNB) are usually resistant due to the outer membrane barrier.

• Cell-Wall Deficient Forms:
      * L-forms: Bacteria that lose their cell wall during the life cycle due to mutations or induction by lysozyme/penicillin. Named after the Lister Institute.
      * Protoplast: A Gram-positive bacterium that has lost its cell wall, leaving only the cell membrane. It is very fragile and easily lysed.
      * Spheroplast: A Gram-negative bacterium that has lost its peptidoglycan but retains its outer membrane. It is weakened but less fragile than a protoplast.

Plasma Membrane and Mesosomes

• Plasma Membrane Structure:
      * Located directly under the cell wall.
      * Very thin lipid bilayer composed of $30-40\%$ phospholipids and $60-70\%$ proteins.
      * Exceptions: $Mycoplasma$ contains sterols; Archaea contain unique branched hydrocarbons.
      * Hopanoids: Synthesized from the same precursors as steroids; they stabilize and modulate the fluidity of the bacterial membrane.

• Plasma Membrane Functions:
      * Transport of ions, nutrients, and waste.
      * Selective permeability.
      * Site for energy functions (respiration and ATP synthesis).
      * Enzyme synthesis for structural macromolecules (cell envelope/appendages).
      * Secretion of toxins and enzymes into the environment.

• Mesosomes:
      * Internal pouches formed by the folding of the cell membrane into the cytoplasm.
      * Prominent in Gram-positive bacteria; smaller and harder to see in Gram-negative.
      * Functions: Cell wall synthesis, guiding duplicated chromosomes during cell division, and increasing surface area for enzymatic activity.

The Glycocalyx

• Definition: Extracellular material secreted by bacterial cells.

• Types:
      * Capsule: Attached tightly to the cell with definite boundaries.
      * Slime Layer: Loosely associated and easily washed off.

• Composition: Usually a layer of polysaccharides, sometimes proteins.
      * Example: $Streptococcus\,pyogenes$ has a hyaluronic acid capsule.
      * Example: $Bacillus\,anthracis$ has a capsule of poly-$D$-glutamic\,acid.

• Functions: Protection, identification, tissue attachment, and acting as an antibiotic barrier.

• Medical Importance: Used for rapid serological identification of pathogens like $Meningococcus$, $Hemophilus\,influenzae$, and $Klebsiella\,pneumoniae$.

• Antigenicity: The capsular antigen is known as the K antigen.

Bacterial Appendages: Pili, Fimbriae, and Flagella

• Pili (Pilus):
      * Found only in Gram-negative bacteria.
      * Tubular, hairlike protein structures; larger and rarer than fimbriae.
      * Attachment Pili: Allow bacteria to attach to other cells.
      * Sex Pili: Used for conjugation (transfer of genetic material).

• Fimbriae:
      * Small in size but large in number.
      * Used for adhesion to cells and surfaces.
      * Responsible for the formation of biofilms.
      * Involved in the pathogenesis of $Gonococcus$ and $E.\,coli$.

• Flagella (Flagellum):
      * Long appendages used for motility and chemotaxis (positive and negative movement relative to chemicals).
      * Found in all spirilla, half of bacilli, and rarely in cocci.
      * Antigenicity: Known as the H antigen, used in serological identification of $Salmonella$.
      * Phylogenetic Arrangements:
          * Monotrichous: Single flagellum at one end (e.g., $Pseudomonas$).
          * Lophotrichous: A tuft of flagella at one end.
          * Amphitrichous: Flagella at both poles (bipolar).
          * Peritrichous: Multiple flagella randomly dispersed around the cell (e.g., $E.\,coli$).
          * Atrichous: No flagella (e.g., $Lactobacilli$).

Flagellar Structure and Mechanism

• Morphological Regions:
      * Helical Filament: Outermost region ($90\%$ of length); made of the protein flagellin arranged in a helix around a hollow core.
      * Hook: Curved area where the filament attaches; made of a different protein.
      * Basal Body: Terminal portion that fixes the flagellum to the cell wall and membrane. Composed of a central rod and rings.

• Gram-Specific Ring Structure:
      * Gram-negative: 2 pairs of rings (Outer pair in outer membrane/peptidoglycan; Inner pair in plasma membrane/SM ring).
      * Gram-positive: Only the inner pair of rings is present.

• Physical Properties:
      * Length: Up to $15\,\mu m$.
      * Rotation: Approximately $20,000\,rpm$.
      * Energy Consumption: Approximately $10^{16}\,W$.
      * Efficiency: Close to $100\%$.
      * Biosynthesis: Self-assembly of about $25$ different proteins.

Bacterial Motility and Detection

• Movement Types:
      * Run or Swim: Movement in one direction for a length of time.
      * Tumble: Periodic, abrupt random changes in direction.
      * Swarming: Rapid wavelike growth across a solid medium.

• Specialized Motility:
      * Axial Filaments: Found in Spirochaetes (e.g., $Treponema\,pallidum$). Periplasmic flagella (fibrils) inside an outer sheath that cause corkscrew-like movement.
      * Gliding Motility: Employed by cyanobacteria, myxobacteria, and cytophagas. No visible external structures; bacteria coast at rates up to $3\,\mu m/s$.

• Detection Methods:
      * Microscopic: Wet mount or hanging drop preparation using dark field microscopy.
      * Macroscopic: Stab inoculation in semisolid media. Motile bacteria show diffuse growth/turbidity away from the line of inoculation; non-motile growth is limited to the stab line.

• Visualization: Flagella are too thin for light microscopy and must be coated with mordants (tannic acid, potassium alum) and stained with basic fuchsin or pararosaniline (Leifson/Gray methods).

Cell Cytoplasm and Nuclear Material

• Cytoplasm:
      * Encased by the cell membrane.
      * Composition: $70-80\%$ water (solvent) mixed with sugars, amino acids, and salts.
      * Function: Primary site for biochemical and synthetic activities.

• Bacterial Chromosome:
      * A single circular strand of DNA.
      * Aggregated in a dense area called the nucleoid.
      * The DNA is tightly coiled into a long molecule.