BIO101

BIO 101 Diversity of Living Organisms Lecture 2: Prokaryotes (Cont’d)

Instructor: Elvis Awuni (PhD)
Department: Biochemistry
School: Biological Sciences
Institution: College of Agric. & Natural Sciences, University of Cape Coast, Cape Coast, Ghana
Contact: elvis.awuni@ucc.edu.gh

Gram Staining Overview

• Definition:
  • Gram stain (Gram staining or Gram’s method) is a technique used by scientists to classify bacteria based on cell wall composition.
• Purpose:
  • Divides most clinically significant bacteria into two main groups, and serves as the initial step in bacterial identification.

Classification Based on Gram Stain

• Gram-Positive Bacteria:

  • Staining Results: Purple/Blue
  • Characteristics:
  • Thick peptidoglycan cell walls.

• Gram-Negative Bacteria:

  • Staining Results: Red/Pink
  • Characteristics:
  • Cell walls contain thin peptidoglycan and lipopolysaccharides.
    • Peptidoglycan Definition:
    • A unique macromolecule composed of repeating frameworks of long polysaccharide (glycan) chains cross-linked by short peptide fragments.
    • Provides structural support to bacterial cell walls, preventing bursting or collapsing due to osmotic pressure changes.

Structure of Cell Walls

Gram-Positive Bacteria Structure

• Components:
  1. Inner plasma membrane
  2. Thick layer of peptidoglycan
  3. Tightly bound acidic polysaccharides including:
  • Teichoic acid
  • Lipoteichoic acid
  1. An outer capsule
• Staining Behavior:
  • Retains crystal violet, appearing purple/blue.

Gram-Negative Bacteria Structure

• Components:
  1. Inner plasma membrane
  2. Thin layer of peptidoglycan
  3. Outer membrane containing lipopolysaccharides (LPS)
  4. An outer capsule
• Staining Behavior:
  • Loses crystal violet, appearing pink/red.

Principle and Procedure for Gram Staining

Steps in Gram Staining

1. Fixation of Sample:

   • Mounting sample to microscope slide through:
     • Heating
     • Using methanol

2. Application of Crystal Violet (Primary Stain):

   • Stains all cells purple/blue.

3. Application of Iodine Solution (Mordant):

   • Forms a crystal violet-iodine (CV-I) complex, all cells still appear blue at this point.

4. Decolorization:

   • Use of organic solvents (e.g., acetone, ethanol) to extract the blue dye complex:
     • Successfully removes dye from gram-negative bacteria to a larger extent than from gram-positive bacteria, resulting in:
     • Colorless gram-negative bacteria
     • Blue/Purple gram-positive bacteria

5. Application of Safranin (Counter Stain):

   • Treats sample with safranin (red dye), coats decolorized gram-negative cells pink/red, while gram-positive bacteria remain purple/blue.

Chemical Interactions in Gram Staining

• Crystal Violet:
  • Dissociates in aqueous solution to produce CV+ and Cl- ions that penetrate bacterial cell walls and membranes, staining cells purple/blue.
• Iodine (I-):
  • Interacts with CV+ to form CV-I complexes in the cytoplasm and outer cell layers.
• Decolorizing Agent (Ethanol):
  • Interacts with membrane lipids of both gram-positive/negative bacteria:
  • In gram-negative bacteria, the outer membrane is lost, exposing the thin peptidoglycan layer.
  • In gram-positive bacteria, alcohol dehydrated the thick peptidoglycan, trapping CV-I complexes within the cell.
  • After decolorization:
  • Gram-positive remains purple/blue.
  • Gram-negative loses color and takes up red from safranin.

Clinical Relevance of Gram Stain

• Antibiotic Targeting:
  • Many antibiotics target peptidoglycan, damaging bacterial cell walls.
• Gram-negative Antibiotic Resistance:
  • Typically more antibiotic-resistant due to their cell wall structure containing a thin layer of peptidoglycan.

Classification of Bacteria

Gram-Positive Bacteria

• Classification Overview:
  • Aerobe or facultative anaerobe: e.g., Staphylococcus, Streptococcus
  • Anaerobe: e.g., Clostridium, Actinomyces
  • Rods: e.g., Bacillus, Corynebacterium

Gram-Negative Bacteria

• Classification Overview:
  • Aerobe: e.g., Neisseria
  • Rods: e.g., Escherichia, Salmonella, Shigella
  • Anaerobe: e.g., Bacteroides, Fusobacterium
  • Others: e.g., Bordetella, Campylobacter

Nitrogen Cycle

• Components:
  • Nitrogen-fixing bacteria in soil and nodules
  • Dead organisms and animal waste
  • Atmospheric nitrogen (N2)
  • Processes:
  • Nitrification
  • Denitrification
  • Nitrogen-fixing

Nitrogen Fixation

• Importance of Nitrogen:
  • Essential for survival and growth; nitrogen is a component of amino acids and nucleotides.
  • Atmospheric nitrogen (N2): Approx. 78% of air, inert due to triple bond (N≡N).
  • Conversion Requirement:
  • Must be converted to ammonia (NH3) or nitrate (NO3) ions for biological use.
• Process of Nitrogen Fixation:
  • Defined as the conversion of atmospheric nitrogen (N2) into inorganic NH3 or NO3.

Main Forms of Nitrogen Fixation

1. Atmospheric Nitrogen Fixation
2. Industrial Nitrogen Fixation
3. Biological Nitrogen Fixation

Biological Nitrogen Fixation

• Process Entrusted to Nitrogen-Fixing Bacteria:
  • Requires complex enzymes and a high expenditure of ATP.
• Symbiotic Relationships:
  • Occur with leguminous (e.g., soybeans) and non-leguminous plants (e.g., alder).
  • Free-living nitrogen-fixing bacteria exist in the soil.
  • Cyanobacteria: Main nitrogen-fixing group in aquatic environments, helping maintain nitrogen balance.

Biological Nitrogen Fixation Reaction

• Reaction Equation:
  (N2 + 8H^+ + 8e^- + 16 ATP ightarrow 2NH3 + H2 + 16ADP + 16Pi)
• Enzyme Complex:
  • Nitrogenase: Comprises two proteins:
  1. Iron-containing protein: Catalyzes ATP breakdown (ATP → ADP + P_i).
  2. Molybdenum-iron containing protein: Converts N2 to NH3.
  • Inhibition:
  • Nitrogenase is inhibited by oxygen.

Examples of Nitrogen Fixing Bacteria

• Free Living Aerobic:

  • Azotobacter, Beijerinckia, Klebsiella, Cyanobacteria (lichens)

• Free Living Anaerobic:

• Associative:

  • Azospirillum

• Symbionts:

  • Rhizobium (legumes), Frankia (alder trees)

Cyanobacteria (Blue-Green Algae/Cyanophyta)

• Characteristics:
  • Photoautotrophic bacteria capable of photosynthesis, producing oxygen gas (O2) as a by-product.
  • Lack chloroplasts but possess thylakoids and contain chlorophyll-a plus other pigments.
  • Some can fix N2 into NH3.
  • Gram-Negative: Most produce toxins.

Morphological Forms of Cyanobacteria

1. Unicellular Forms
2. Colony-Forming Forms
3. Filamentous Forms without Heterocyst Formation
4. Filamentous Forms with Heterocyst Formation:
   • Involved in nitrogen fixation.

Nitrogen Fixation in Cyanobacteria

• Process Triggered by Nitrogen Starvation:
  • Formation of heterocyst:
    • Enlarged cell with a thick three-layered wall, permeable to N2 but impermeable to O2.
    • Photosynthesis is halted to prevent O2 production (which inhibits nitrogenase).

Role of Heterocyst in Nitrogen Fixation

• Heterocyst Functionality:
  1. Thick cell wall formation (three layers) to create anaerobic conditions.
  2. Degradation of photosystem II (prevents O2 production).
  3. Producing oxygen-scavenging species.

Economic Importance of Prokaryotes

• Decomposers:

  • Help maintain environmental equilibrium by decomposing dead organisms.
  • Examples: Bacillus subtilis, Pseudomonas fluorescens.

• Producers:

  • Photosynthetic prokaryotes are vital producers in food chains.
  • Examples: Cyanobacteria, green/purple sulfur bacteria.

• Nitrogen Fixers:

  • Provide 90% of nitrogen utilized by other organisms.
  • Examples: Cyanobacteria, Desulfovibrio, Azospirillum, Rhizobium.

Benefits of Prokaryotes to Humans

• Food Production:
  • Some bacteria are essential in producing various foods (e.g., cheese, yogurt).
• Water Treatment:
  • Certain bacteria digest petroleum and remove pollutants from water.
• Drug Production:
  • Bacteria used in genetic engineering to synthesize drugs.

Prokaryotes in Research and Technology

• Genetic Technology Applications:
  • E. coli used for gene cloning.
  • Agrobacterium tumefaciens utilized for producing transgenic plants.
• Natural Plastics Production:
  • Certain bacteria can produce biodegradable natural plastics.

Harmful Effects of Prokaryotes

• Pathogenic Prokaryotes:
  • Known pathogenic prokaryotes are primarily bacteria, causing approximately half of human diseases.
• **Mechanisms of Disease:
  • Some bacteria destroy living tissue for sustenance; others produce toxins harmful to living tissues.
• Interference with Normal Functioning:
  • Disease occurs when bacteria disrupt the normal functioning of organisms.

Examples of Diseases Caused by Bacteria

Prevention and Treatment of Bacterial Infections

Antibiotics

• Definition:
  • 'Anti' means 'against', 'bio' means 'life', thus antibiotics kill living organisms, rendering them ineffective against viruses (which are non-living).
• Mechanism of Action:
  • Kill bacteria by:
  1. Blocking growth
  2. Blocking reproduction
  3. Inhibiting essential life processes

Common Antibiotics

Antibiotic Resistance

• Global Problem Factors:
  • Over-prescription of antibiotics
  • Over-the-counter availability without restrictions
  • Lack of new antibiotics
  • Patients not completing antibiotic courses

Bacterial Vaccines

• Definition:
  • Killed or inactivated (attenuated) bacteria that activate the immune system to prevent and combat bacterial infections.
• Examples of Available Vaccines:
  • Vaccines against tuberculosis, tetanus, cholera, etc.