Introduction to Microbial Physiology and the Escherichia coli Paradigm

Introduction to Microbial Physiology: The Escherichia coli Paradigm

• Definition and Scope: Microbial physiology is a vast discipline that covers the study of thousands of unique microorganisms. Due to the scale of the field, a solid foundation is built using representative organisms.
• The Paradigm Organism: The gram-negative organism Escherichia coli is used as the primary paradigm in this text. It serves as a central model for understanding microbial physiology and genetics.
• Alternative Strategies: While E. coli is the focus, other organisms are included to provide counterexamples or illustrate alternative biochemical strategies for achieving similar physiological goals.
• Conceptual Objective: This introductory chapter provides a broad portrait of the microbial cell, serving as a point of confluence where learners can see the interrelations between different aspects of physiology.

General Bacterial Cell Structure

• Basic Morphologies: Bacteria commonly exist in three fundamental shapes:     * Spherical: Known as coccus.     * Rod-shaped: Known as bacillus.     * Spiral: Known as spirillum.
• Specialized Forms: Beyond basic shapes, bacteria can be specialized as budding, sheathed, or mycelial.
• Prokaryotic Nature: Bacteria are classified as prokaryotic because they do not possess a membrane-bound nucleus, distinguishing them from eukaryotic microorganisms.
• Quantitative Composition of a "Typical" Cell (E. coli):     * Proteins: Approximately $1000 - 2000$ different proteins, totaling circa $10^6$ molecules per cell.     * tRNAs: Roughly $60$ different types of Transfer RNA, totaling circa $10^6$ molecules per cell.     * Storage Materials: Includes glycogen.     * Genetic Machinery: Includes the chromosome, RNA polymerase, RNA, and polyribosomes.     * Coupled Processes: Bacteria exhibit coupled transcription and translation.

The Bacterial Cell Surface and Wall

• Functional Interface: The cell surface serves as the interface between the cell and its environment. Its primary roles include:     * Protecting the cell interior from external hazards.     * Maintaining the integrity of the cell as a discrete entity.     * Enabling transport of large molecules (e.g., glucose, vitamin $B_{12}$, amino acids, nucleosides, and exported proteins).
• The Gram Stain (1884): Devised by Danish investigator Christian Gram. The process involves:     * Staining with crystal violet dye.     * Decoloration with $95\%$ ethanol.     * Gram-positive: Cells that retain the crystal violet stain.     * Gram-negative: Cells that lose the stain during decoloration.
• Composition of Peptidoglycan:     * A linear polymer of alternating units of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM).     * A short peptide chain is attached to the muramic acid (NAM) unit.     * Peptide Cross-bridging: A common feature where peptide chains are linked.         * In gram-positive Staphylococcus aureus, cross-bridging is close to $100\%$.         * In gram-negative Escherichia coli, cross-bridging may be as low as $30\%$.
• Gram-Positive Cell Surface Characteristics:     * Composed of two major structures: the cell wall and the cell membrane.     * The wall consists of multiple layers of peptidoglycan.     * Lipoteichoic Acid: Synthesized at the membrane surface, it is only present in gram-positive organisms and may extend through the peptidoglycan layers to the outer surface.
• Gram-Negative Cell Surface Characteristics:     * The peptidoglycan layer is generally a single monolayer.     * Outer Membrane: Surrounds the cell; composed of phospholipids, lipopolysaccharides (LPS), enzymes, and proteins (including lipoproteins).     * Periplasmic Space: The region between the inner (cytoplasmic) membrane and the outer membrane. It is a periplasmic gel traversed by various enzymes and proteins.

Membranes and Specialized Components

• Cytoplasmic Membrane (Inner Membrane):     * Shared by both gram-positive and gram-negative cells.     * Structure: A lipid bilayer composed of phospholipids, glycolipids, and diverse proteins.     * Function: Proteins provide structural support or act as transporters for sugars, amino acids, and other metabolites.
• Outer Membrane Features of Gram-Negative Cells:     * O Antigens: Formed by external polysaccharide chains of the lipopolysaccharide (LPS). Used for identification.     * Endotoxin Activity: The lipid-containing component of LPS is responsible for the shock observed in severe gram-negative infections.     * Porins: Protein triplets that form pores in the outer membrane, permitting the passage of large molecules into the periplasmic space.
• Bacteriophage Receptors: Cell surfaces contain specific carbohydrate or protein receptor sites that bacteriophages (viruses that infect bacteria) use for attachment and subsequent cell invasion.
• Transport Comparison:     * Gram-positive: The cytoplasmic membrane has immediate access to external media components.     * Gram-negative: Nutrients must first traverse the outer membrane (via porins) before reaching the cytoplasmic membrane for transport into the cytosol.

Capsules and External Appendages

• Capsules (Slime Layers): Material produced external to the cell wall.     * Composition: Polysaccharides (carbohydrate polymers) or polypeptides (amino acid polymers, often using D-isomers).     * Streptococcus pneumoniae Type III: Capsule consists of glucose and glucuronic acid in alternating $\beta-1, 3-$ and $\beta-1, 4-$ linkages. This is essential for virulence.     * Bacillus anthracis: Produces a polypeptide capsule made of D-glutamic acid subunits, which is a key virulence factor.
• Flagella (Organs of Locomotion):     * Enable motility in aqueous environments.     * Structure: Simple strands of the protein flagellin woven into helical organelles.     * Basal Body: Attaches the flagellum to the cell surface and contains a motor that turns the flagellum to propel the organism.
• Pili or Fimbriae:     * Shorter and more rigid than flagella.     * Origin: Arise from a basal body or granule located in or just beneath the cytoplasmic membrane.     * Common Pili: Play a role in cellular adhesion to surfaces or host cells.

Ribosomes and Protein Synthesis

• Appearance: Fine granular particles in the cytoplasm.
• Composition: Approximately $65\% \text{ RNA}$ and $35\% \text{ protein}$.
• Function: Orchestrate the polymerization of amino acids into proteins.
• Polyribosomes (Polysomes): Chains of ribosomes held together on a single messenger RNA (mRNA) molecule.
• Sedimentation and Svedberg Coefficients ($S$):     * $S$ denotes the rate of sedimentation of a macromolecule in a centrifugal field, related to molecular size.     * Prokaryotic Ribosome: Total coefficient of $70S$. It consists of two subunits: a $50S$ unit and a $30S$ unit.     * Eukaryotic Ribosome: Total coefficient of $80S$. It consists of a $60S$ and a $40S$ subunit. Generally associated with the endoplasmic reticulum.
• Mitochondrial Ribosomes: Curiously, eukaryotic mitochondria contain $70S$ ribosomes. This supports the endosymbiotic theory that mitochondria evolved from endosymbiotic prokaryotic cells.

Synthesis of DNA, RNA, and Protein

• The E. coli Chromosome: A single, circular, double-stranded DNA (dsDNA) molecule. It contains the nucleotide sequence for all information required for structural growth.
• Binary Fission: The process of bacterial replication resulting in two daughter cells.     * Triggered when the cell reaches a specific mass-to-DNA ratio.     * Involves DNA replication followed by the construction of a centrally located cross-wall.
• DNA Replication Mechanism:     * Origin: The specific starting point where the dsDNA unwinds.     * Daughter Strands: New DNA is synthesized opposite each parent strand.     * Enzymatic Activity: DNA polymerase uses the parent strand as a template.     * Base Pairing Rule: Adenine ($A$) resides opposite thymine ($T$); cytosine ($C$) resides opposite guanine ($G$).     * Directionality: Replication proceeds in both directions from the origin until the replication forks meet at the terminus, which is situated $180^\circ$ from the origin.     * Components involved: Unwinding enzyme, RNA primer, leading strand, lagging strand (featuring Okazaki fragments), and DNA polymerase.