Microbiology Chapter on Gene Transfer

Microbial Classification

• Microorganisms are divided into eukaryotes, which contain a membrane-bound nucleus, and prokaryotes, which do not.
• Eukaryotic cells have a true nucleus with a nucleolus and nuclear membrane. Prokaryotic cells have a premature nucleus without a nucleolus or nuclear membrane.
• Eukaryotic chromosomes are more than one. Prokaryotic chromosomes are a single, double-stranded DNA molecule.
• Eukaryotic cytoplasmic membranes contain sterols. Prokaryotic cytoplasmic membranes generally do not contain sterols, except in mycoplasmas.
• Eukaryotes have 80S ribosomes. Prokaryotes have 70S ribosomes.
• Eukaryotic respiratory systems are located in mitochondria. Prokaryotic respiratory systems are located in the cytoplasmic membrane.
• Eukaryotes multiply by mitosis. Prokaryotes multiply by binary fission.
• Examples of eukaryotes include fungi. Examples of prokaryotes include bacteria and rickettsia.

Morphology of the Bacterial Cell

• Bacteria are small, unicellular prokaryotic organisms with rigid cell walls.
• They multiply by simple binary fission.
• Gram staining divides bacteria into two groups: Gram-positive and Gram-negative.
  • Gram-positive bacteria resist decolorization by alcohol, appearing violet under a microscope.
  • Gram-negative bacteria are decolorized by alcohol, appearing red under a microscope.
• Bacterial cell walls have a peptidoglycan layer.
  • Gram-positive cell walls have a thick peptidoglycan layer (50-60% of the cell wall). They often have teichoic acid.
  • Gram-negative cell walls have a thin peptidoglycan layer (5-10% of the cell wall). They have an outer membrane, lipopolysaccharide layer, and periplasmic space.

Morphology of Bacterial Cell: Cell Wall Components

• Gram-positive cell wall:
  • Peptidoglycan layer: 50-60% of the cell wall thickness.
  • Teichoic acid: Antigenic structure.
• Gram-negative cell wall:
  • Peptidoglycan layer: 5-10% of the cell wall thickness
  • Lipoprotein layer
  • Outer membrane: outside the lipoprotein layer.
  • Lipopolysaccharide (LPS) layer: endotoxin (lipid A) + polysaccharide O antigen
  • Periplasmic space

Function of Bacterial Cell Wall

• Preservation of cell shape (rigidity)
• Osmotic sensitivity.
• Antigenicity
• Toxicity (LPS in Gram-negative bacteria).
• Target for antibiotics (e.g., penicillin and cephalosporins).

Morphology of Bacterial Cell: Other Components

• Cytoplasmic membrane (Plasma membrane):
  • Semi-permeable, double layered structure. Composed of phospholipids and proteins.
• Function: Selective permeability and active transport; energy production, site of respiration; excretion of pathogenic proteins and toxins.
• Mesosome: Irregular, convoluted invagination of cytoplasmic membrane; sites of respiratory enzymes; increased membrane surface area; involvement in cell division.

The Bacterial Genome

• Nuclear body: DNA concentrated in the cytoplasm within a nucleoid; lacks a nuclear membrane or nucleolus.
• Plasmid: Extra-chromosomal DNA molecule.
• Ribosomes: 70S; comprised of 30S and 50S subunits. Protein synthesis.
• Target of antibiotics: Tetracycline and chloramphenicol.

Extracellular Structures and Appendages

• Capsule: Polysaccharide layer (in most bacteria but not Bacillus anthracis which is polypeptide). Virulence factor against phagocytosis; antigenic properties; adherence of bacteria to tissues.
• Flagella: Filamentous appendages enabling movement; motility; antigenicity.
• Pili (fimbriae): Short, hair-like appendages; adhesion; virulence factors; important in bacterial identification (antigenic).

Bacterial Endospores

• Highly resistant, resting structures.
• Formed by some Gram-positive bacilli in adverse environmental conditions (depletion of nutrients, heat, dryness etc).
• Core with essential enzymes and thick peptidoglycan cortex (spore coat).
• Resistant to heat, chemicals, and radiation.
• Important medically due to resistance to heating and need to sterilize materials by autoclaving.
• Can be used for contamination testing in wounds or soil samples.

Bacterial Growth Requirements

• Bacterial growth is increase in cell mass.
• Nutrition
  • Autotrophic: Utilize inorganic sources of carbon and nitrogen.
  • Heterotrophic: Require organic carbon and nitrogen sources (like sugar and proteins).
• Gaseous requirements (e.g. Oxygen)
  • Strict aerobic: Require oxygen for metabolism

Bacterial Products

• Endopigments: Localized in the bacteria; color the bacterial colonies (e.g., staphylococcus aureus is golden).
• Exopigments: Diffuse out of bacteria into surrounding medium; color the medium e.g., pseudomonas is greenish blue; some have roles in bacterial respiration

Bacterial Toxins

• Exotoxins: Diffusible toxins; protein nature; highly toxic; strong antigenic specificity (target specific cells and tissues); heat labile.
• Endotoxins: Bound to the body and released only when the organism disintegrates; lipopolysaccharide nature; less toxic; weak antigenic specificity (non-specific); heat stable

Bacterial Growth and Reproduction

• Bacterial Growth = increase in number via asexual binary fission.
• The bacterial growth curve has lag, log (exponential), stationary and death (decline) phases.
• Lag, exponential, stationary and death phases have different properties depending on characteristics like type of organism, initial inoculum size, and medium.

Bacterial Genetics

• Bacteria are convenient for genetic studies due to haploid nature, rapid growth, and large numbers.
• Chromosome: major DNA molecule; circular.
• Extra-chromosomal DNA: Plasmid; circular DNA molecule capable of autonomous replication; often carries non-essential genes; can be transferred between cells; may confer virulence or antibiotic resistance.
• Transposons: Jumping genetic elements; can move between different locations within the genome; carry genes for antibiotic resistance and other traits. Can also cause mutations.

Bacteriophages

• Bacteriophage: Virus that infects bacteria; lytic cycle or lysogenic cycle; can be used in research and treatment.

Antimicrobial Chemotherapy

• Bactericidal drugs: rapid killing of bacteria; irreversible; examples are penicillin, cephalosporins and aminoglycosides.
• Bacteriostatic drugs: inhibit bacterial multiplication; reversible; host's immune system is required to eliminate the bacteria; includes sulfonamides, tetracyclines and chloramphenicol.
• Mechanisms of action: Inhibition of bacterial cell wall synthesis, cytoplasmic membrane functions, protein synthesis, nucleic acid synthesis; competitive inhibition.

Resistance to Antimicrobial Agents

• Several mechanisms lead to resistance: inactivation of enzyme by production; alteration of permeability to drug; alteration of target, alteration metabolic pathway, and alteration of enzyme.

Pathogenicity and Infectious Disease

• Pathogenicity: ability of organism to cause disease.
• Virulence: degree of pathogenicity.
• Infection: multiplication of infectious agent within body, may be inapparent or asymptomatic.
• Disease: development of signs and symptoms of disease.
• Sources of infection: human, animal, or inanimate sources (e.g., soil).
• Transmission: direct (contact, spread of large droplets), indirect (vector borne, airborne), or through inanimate sources.

Antimicrobial prophylaxis

• Prophylaxis: Measures to prevent infection in situations where risk of infection is high.
• Combining antimicrobial drugs can be synergistic, allowing for more effective treatment or to prevent the rapid development of antibiotic resistance.

Genetic Engineering and Recombinant DNA Technology

• Recombinant DNA technology is used to isolate genes, create new combinations, and clone genes using vectors (e.g. plasmids or bacteriophages). This has numerous applications including, vaccine production, production of essential proteins and molecular diagnosis.

Test your knowledge on gene transfer mechanisms in microbiology with this quiz. It covers key concepts, including transformation, transduction, and recombinant DNA technology. Understand the processes that enable genetic recombination and the implications for gene therapy.