Prokaryotic Cell

Learning Outcomes

1. Differentiate between Prokaryotes and Eukaryotes

2. Identify clinically important bacteria based on microscopic appearance

3. Define the role of certain cellular structures in the pathogenesis of infection

4. Describe the processes involved in bacterial growth

5. Describe the structure of bacterial DNA and the process of DNA replication

6. Explain bacterial gene expression (transcription and translation)

7. Describe the clinical significance of plasmids and DNA mutation

1. Differentiate between Prokaryotes and Eukaryotes

• Prokaryotes

  • No nucleus

  • Contains a cell wall

  • Lacks cell organelles

• Eukaryotes

  • Contains a nucleus

  • No cell wall

  • Has cell organelles such as mitochondria, chloroplasts, endoplasmic reticulum

2. Identify Clinically Important Bacteria Based on Microscopic Appearance

Microscopy

• Allows examination of

  • Bacterial cell shape

  • Colour (due to staining)

  • Size

The Gram Stain

• Most important differential staining method in microbiology and consists of four steps:

  1. Crystal violet

  • Primary stain added to specimen smear.

  1. Iodine

  • Acts as a mordant that makes the dye less soluble, so it adheres to cell walls.

  1. Alcohol

  • Used as a decolorizer that washes away stain from gram-negative cell walls.

  1. Safranin

  • Counterstain that allows dye to adhere to gram-negative cell walls.

• Gram(+): appears purple

• Gram(-): appears red

Staining Characteristics of Bacteria

• Crystal violet-iodine complex forms inside cells (blue color).

• Alcohol treatment

  • Gram-negative: High lipid content in cell envelope extracted, making it permeable.

  • Gram-positive: Low lipid content, dehydrated by alcohol, making it impermeable.

• Mycobacteria: Stained using Ziehl-Neelsen stain due to high wax content in their cell envelope.

• Mycoplasmas: The smallest known bacteria, no cell wall to stain.

• Yeasts: Can be Gram stained even though they are not classified as Gram+ or Gram-.

  • Candida albicans can retain crystal violet color after Gram stain.

  • Mycobacterium tuberculosis is acid-fast, appears red due to resistance to decolorizing with acid, retains carbol fushin dye.

3. Define the Role of Cellular Structures in the Pathogenesis of Infection

• Cell Appendages:

  • Flagella: Motility and chemotaxis

  • Pili: Adherence to surfaces

• Spores:

  • Some Gram-positive bacteria can form spores for protection under adverse conditions.

  • Gram-negative bacteria cannot form spores.

• Capsules and Slime Layers:

  • Surround many bacterial cells, provide protection from phagocytosis and antibiotics, and play a role in bacterial adherence.

• Clinical Significance of Biofilms:

  • Biofilms complicate treatment. Examples include Pseudomonas aeruginosa (seen in cystic fibrosis patients) and Staphylococcus epidermidis (catheter-related infections).

4. Describe the Processes Involved in Bacterial Growth

Bacterial Growth in Laboratory Settings

• Growth mediums include liquid broths and nutrient agar plates

Bacterial Division

• Binary Fission:

  • Chromosome divides to produce two identical copies.

  • Enables rapid growth and adaptation under selective pressure which can lead to mutations.

• Growth Requirements:

  • Energy from enzymatic breakdown of organic substrates (catabolism of carbohydrates, lipids, proteins)

  • Nutritional requirements:

  • Water

  • Carbon

  • Nitrogen

  • Inorganic salts

  • Iron

  • Environmental conditions:

  • Temperature

  • pH

  • Availability of O₂ (aerobic vs anaerobic)

5. Describe the Structure of Bacterial DNA and the Process of DNA Replication

Bacterial Genome

• Total collection of genes including on chromosomes and plasmids

• Approximately 4000 genes and 5 million DNA base pairs, organized as a circular molecule of double-stranded DNA (helix).

DNA Structure

• Composed of nucleotides

  • Bases: Guanine (G), Adenine (A), Cytosine (C), Thymine (T)

  • Sugar: deoxyribose

  • Phosphate groups

• The double helix structure stores genetic information necessary for all cellular processes.

Supercoiling

• DNA gyrase: Type II topoisomerase that catalyzes negative supercoiling, releasing tension for replication and transcription.

DNA Replication Process

• Semi-conservative replication consisting of four steps:

  • Initiation: Begins at the origin of replication (oriC), where helicase unwinds dsDNA.

  • Elongation:

  • DNA polymerase attaches and synthesizes new strands in the 5’ to 3’ direction.

  • RNA primase lays down an RNA primer, creating Okazaki fragments on the lagging strand.

  • Okazaki Fragments: Linked by DNA ligase.

  • Proofreading: Errors corrected by DNA polymerase; mutation rates between 10^{-3} to 10^{-9} per cell division.

  • Termination: Completion results in two identical daughter helices.

6. Explain Bacterial Gene Expression (Transcription and Translation)

• Gene Structure:

  • Genes include the sequences of DNA encoding proteins, occurring individually or in operons (rare in eukaryotes).

• Transcription:

  • Occurs at the promoter region; RNA polymerase synthesizes mRNA from the DNA template.

  • In RNA, thymine is replaced by uracil.

• Translation:

  • mRNA is translated by ribosomes, directed by tRNA that carries amino acids for protein synthesis.

7. Describe the Clinical Significance of Plasmids and DNA Mutation

Plasmids

• Small, circular, extrachromosomal DNA that replicates independently and can be transferred between cells.

• Often contain genes for

  • Antibiotic resistance

  • Virulence factors (toxins, etc.)

  • Metabolic functions

Gene Mutations

• Common sources of genetic variation may be spontaneous or induced (mutagens), with rates from 10^{-3} to 10^{-9} mutations per division.

• Types of Mutations:

  • Substitution: Change in one nucleotide, can result in silent mutations (no change in protein functionality) or significant alterations.

  • Deletions: Can cause frameshift mutations that alter the reading frame, affecting the coding downstream.

  • Insertions: Frameshift mutations often lead to incorrect protein lengths, occurring due to premature stop codons.

Summary

• Clinically significant structural and functional differences exist between Gram-positive and Gram-negative bacteria.

• Understanding bacterial structures, genetic mechanisms, and their roles in infections helps inform treatment strategies, especially concerning antibiotic resistance and evolving virulence.

• Recognition of the importance of plasmids in conferring resistance and the impact of mutations on genetic variation is crucial in clinical microbiology.