Microbiology: Genetic Mutations, Recombinant DNA, and Antimicrobial Therapy Study Notes

Translation Mechanisms and the Role of Transfer RNA (tRNA)

• The Translation Process: The ribosome scans the messenger RNA (mRNA) codon sequence to build a protein chain. It sends a chemical signal, triggering the entry of transfer RNA (tRNA).

• Anticodon Match: The tRNA binds its anticodon sequence to the mRNA codon sequence. Translation ensures accuracy via base-pairing rules; if the sequences match, the amino acid is added to the chain; if not, the tRNA leaves without depositing an amino acid.

• Amino Acid Sequence: The transcript uses phenylalanine ($PHE$) and leucine as examples. A codon like $UUU$ might code for phenylalanine, while $CUK$ (hypothetical placeholder) or $UUA$ may code for leucine.

• Proofreading Mechanism: tRNA acts as a proofreading mechanism to ensure the correct amino acid is placed in the correct sequence.

• Role of tRNA: Its primary function is to transport amino acids from the cytoplasm to the ribosome to build the amino acid chain/protein.

• End of Translation: When the ribosome reaches a stop codon, it detaches from the mRNA, and the process terminates.

Genetic Mutations in Bacterial Cells

• Definition of Mutation: A mutation is any change in the nucleotide sequence of DNA (e.g., $AGC$ changing to $AGG$).

• Frequency and Impact: DNA mutations occur frequently in both human and bacterial DNA. However, much of the DNA is non-coding (filler), so mutations often have no observable effect unless they occur in "gene regions" (the recipes for proteins).

• Phenotypic Changes: Mutations can cause bacteria to look or behave differently, including:

  • Shape: Moving from coccus (spherical) to bacillus (rod-shaped).

  • Arrangement and Size.

  • Antibiotic Resistance: Developing mechanisms to survive medication.

  • Environmental Preferences: Changes in preferred temperature or $pH$.

  • Cell Death: If a mutation occurs in a vital gene region, the cell may be unable to sustain life.

• Growth and Replication: The more a bacterial cell grows, the more replication cycles it undergoes, increasing the probability of making a mistake where the wrong nucleotide (e.g., $T$ instead of $G$) is inserted.

Categories and Mechanics of DNA Mutations

• Spontaneous Mutation:

  • These occur without external influence due to errors in DNA replication machinery.

  • DNA Polymerase III: This is the primary enzyme responsible for synthesizing the DNA chain. It is extremely fast; in organisms like $E. coli$, it can lay down approximately $200$ nucleotides per second ($200\,nucleotides/s$).

  • While it has some self-correcting abilities, it occasionally leaves errors (e.g., substituting a $C$ for an $A$) that become permanent mutations.

• Induced Mutation:

  • These result from exposure to environmental factors known as mutagens.

  • Radiation: Ultraviolet (UV) radiation is a common mutagen used for sterilization.

  • Case Study: Lab Safety Cabinet: A white file cabinet used for safety glasses ostensibly uses UV light bulbs to sterilize equipment by causing rapid, lethal mutations in any present bacteria. Note: The speaker mentions their specific lab cabinet has not functioned for at least $12$ years, requiring the use of alcohol swabs instead.

Specific Types of DNA Mutations

• Point Mutation:

  • A single nucleotide is replaced by another (e.g., $G$ swapped for $T$). The total number of nucleotides remains the same.

  • Wiggle Room (Redundancy): Because multiple codons can code for the same amino acid (e.g., six different sequences for leucine), a point mutation may not change the resulting protein. This is a "silent" effect.

  • Example Case: Changing $AAA$ (codons to $UUU$ for phenylalanine) to $AAT$ (codons to $UUA$ for leucine) alters the protein's function. Conversely, changing $AAA$ to $AAG$ (codons to $UUC$) still results in phenylalanine, causing no change in the final protein.

• Frameshift Mutations:

  • Generally more dangerous/detrimental than point mutations because they alter the "reading frame" for the entire sequence following the mutation.

  • Insertion: An extra nucleotide is added to the DNA code.

  • Deletion: An entire nucleotide is removed from the DNA code.

  • Because the ribosome reads in three-nucleotide intervals (codons), adding or removing one nucleotide shifts every subsequent codon, typically resulting in a non-functional protein and potentially premature stop codons.

• Identification Tip: To identify a mutation type without line-by-line comparison, count the nucleotides. If the count is the same, it is a point mutation. If the count is $+1$, it is an insertion; if $-1$, it is a deletion.

Genetic Engineering and Recombinant DNA Technology

• Definition: The manipulation of DNA to make an organism behave or look in a desired way.

• Microbial Utility: Genetically modifying bacteria to produce human proteins (e.g., insulin, progesterone, estrogen, testosterone).

• Recombinant DNA: The combination of DNA from two different organisms (e.g., inserting a human gene into a bacterial cell).

• Essential Supplies:

  • DNA of Interest: The specific human gene region (e.g., the insulin recipe).

  • Bacterial Plasmid: The circular, advantageous genetic material of the bacteria.

  • Restriction Endonuclease Enzyme: Often called "bacterial scissors." It cuts DNA at specific sequences. Bacteria naturally use this to defend against bacteriophages. It creates "sticky ends" (single-stranded overhangs that seek matches).

  • Ligase: The "glue" enzyme that fuses human DNA fragments and bacterial plasmids together.

  • Host Cell: Frequently $E. coli$ is used due to its rapid binary fission cycle of approximately $20\,min$.

• Process Overview: Extract human DNA $\rightarrow$ Cut with restriction enzyme $\rightarrow$ Open bacterial plasmid with same enzyme $\rightarrow$ Match sticky ends $\rightarrow$ Fuse with ligase $\rightarrow$ Insert into $E. coli$ $\rightarrow$ Bacteria replicate and excrete the desired human protein for medical collection.

Forensic and Diagnostic Applications

• Forensics and Paternity: Because filler DNA varies between individuals, restriction enzymes cut everyone's DNA into different fragment sizes.

• Gel Electrophoresis: A machine that separates DNA segments by size, creating unique "banding" patterns. Matching these patterns can identify suspects or biological fathers.

• Polymerase Chain Reaction (PCR):

  • Described as "in vitro replication" (replication in a lab setting rather than a cell).

  • Sensitivity: PCR is highly sensitive because it amplifies (mass-copies) small amounts of genetic material.

  • COVID-19 Application: Rapid antigen tests often give false negatives if the viral load is low. A PCR test is more reliable because it can take a tiny sample of viral DNA/RNA and amplify it until it is detectable.

Principles of Antimicrobial Therapy

• Goal: To destroy infectious microorganisms without damaging the human host cells (Selective Toxicity).

• Natural Origins: Antibiotics are chemicals naturally produced by bacteria and fungi as defense mechanisms to eliminate competitors for nutrients.

  • Streptomyces (Bacteria): Produces Streptomycin.

  • Penicillium (Fungi/Mold): Produces Penicillin.

• History: We refine these natural enzymes through chemical modification to create drugs like methicillin and amoxicillin.

Drug Susceptibility Testing

• Kirby-Bauer Test:

  • Creates a "bacterial lawn" on an agar plate. Antibiotic discs are placed on top.

  • Zone of Inhibition: The clear area where narrow/broad-spectrum drugs stopped bacterial growth.

  • Measurement: Always measured in millimeters ($mm$) across the diameter.

  • Interpretation Levels (Example: Streptomycin):

    • Susceptible (Sensitive): $\ge 15\,mm$. The drug is effective.

    • Intermediate: $12\text{--}14\,mm$. Drug might work at higher doses/longer durations.

    • Resistant: $\le 11\,mm$ (or $0\,mm$). The drug is ineffective; bacteria survive.

• Tube Dilution Test:

  • Used to find the MIC (Minimum Inhibitory Concentration): the lowest concentration of a drug that still inhibits bacterial growth.

  • Involves a series of tubes with decreasing concentrations of the drug ($100\%$, $75\%$, $50\%$, $25\%$, $0\%$). The first clear tube in the sequence (no turbidity) is the MIC.

• Serum Killing Power Test:

  • Matches the lab setting to the human body (In Vivo test).

  • Takes a blood draw from a patient already on an antibiotic, adds the bacteria to the blood, and checks if the bacteria can survive. If they survive, the drug is being deactivated by proteins in the patient's blood.

Pharmacology and Spectrum of Activity

• Dosage Levels:

  • Toxic Dosage Level: The concentration of the drug that becomes harmful to the host (e.g., causing shock due to rapid toxin release from dying bacteria).

  • Therapeutic Dosage Level: The concentration required to eliminate the pathogen if maintained over a full course (usually $7\text{--}14\,days$).

• Chemotherapeutic Index:

  • $\text{Chemotherapeutic Index} = \frac{\text{Maximum Tolerable Dose}}{\text{Minimum Curative Dose}}$

  • Rule: The higher the index, the safer the drug.

• Spectrum of Activity:

  • Broad Spectrum: Targets a wide range (Gram-positive and Gram-negative). Useful when the causative agent is unknown. Con: Wipes out normal biota (healthy bacteria).

  • Narrow Spectrum: Targets specific bacteria (e.g., Isoniazid for Tuberculosis). Pro: Less damage to normal biota. Con: Identity of the pathogen must be known first.

Superinfections and Prevention

• Definition: A secondary infection that occurs after a broad-spectrum antibiotic wipes out normal biota, allowing resistant pathogens to overgrow.

• Yeast Infection: Broad-spectrum drugs kill Lactobacilli in the vagina, which normally maintain an acidic environment. Without Lactobacilli, yeast grows out of control.

• C. diff (Clostridium difficile):

  • Broad-spectrum drugs kill normal gut flora, allowing C. diff (which many people carry in small amounts) to thrive.

  • Causes Antibiotic-Associated Colitis (diarrhea, cramping, vomiting).

  • Highly contagious via endospores in fecal matter.

• Prevention: The use of Probiotics (ingesting living beneficial microorganisms) like yogurt or supplements to replenish normal biota during and after antibiotic courses.