MICROBIO LECTURE 9 Notes

Mutant Isolation Methods

• Two classic strategies for pulling mutants out of a mixed bacterial population

  • Negative (indirect) selection

  • Start with a master plate containing wild-type colonies.

  • Use a sterile velvet “stamp” to replica-plate onto two new plates:

    1. A fully supplemented control plate.

    2. A plate missing a key nutrient (e.g. histidine).

  • After incubation compare colony patterns.

    • A spot that grows on the complete medium but is absent on the deficient medium identifies a nutritional mutant (auxotroph).

  • “Negative” because you look for what fails to grow.

  • Positive (direct) selection

  • Same velvet-replica set-up, but the test plate contains an inhibitor/antibiotic.

  • Colonies that grow on the antibiotic plate are mutants with a selectable advantage (e.g. drug resistance).

  • Control (non-antibiotic) plate verifies viability of the parental spot.

Terminology Refresher

• Wild type (WT) = naturally occurring strain; usually a prototroph (synthesises all needed metabolites).

• Auxotroph = mutant unable to make an essential molecule; requires supplementation.

The Ames Test (Detecting Chemical Mutagens/Carcinogens)

• Uses an auxotrophic reporter strain (commonly Salmonella His–).

• Procedure

  • Mix bacteria with the suspected mutagen plus rat-liver extract (simulates metabolic activation during digestion).

  • Plate onto agar lacking the missing nutrient (histidine).

  • Revertant colonies (His+) indicate mutagen-induced back-mutation.

• Interpretation: Many revertants ⇒ chemical is likely mutagenic/carcinogenic; follow-up with higher-level models.

Horizontal (Lateral) Gene Transfer in Bacteria

• Genetic material introduced from another cell, not parental lineage.

• Incoming DNA either

  1. Remains extrachromosomal as a plasmid (must carry its own origin, promoter, etc.).

  2. Integrates into the chromosome if homology allows.

Three Main Mechanisms

1. Transformation (DNA-mediated)

• Uptake of “naked” DNA from environment.

• Outcomes

  • Plasmid uptake – usually successful ⇨ new traits immediately express.

  • Linear fragment – needs homologous recombination; most fragments are degraded.

• Competence

  • Species-specific propensity to take DNA.

  • Example: Streptococcus pneumoniae is species-restricted; E. coli is highly promiscuous (research work-horse).

• Widely exploited in genetic engineering.

2. Conjugation

• Requires a conjugative plasmid (F plasmid prototype).

• Encodes a type IV sex pilus.

  • Gram-negative: pilus can span a gap.

  • Gram-positive: cells draw close, fuse walls, then transfer DNA.

• Plasmid is copied during transfer ⇒ both donor (F+) and recipient become F+.

• Cargo genes

  • Antibiotic resistance (R-plasmids).

  • Virulence factors, toxin genes, metabolic pathways.

• Plasmid incompatibility: a cell cannot stably maintain two plasmids using the same origin/promoter—one will be lost.

3. Transduction (phage-mediated)

• Bacteriophages accidentally package host DNA and deliver it to new cells.

  • Generalized transduction: lytic phage randomly incorporates any host fragment.

  • Specialized transduction: temperate phage excises imprecisely, carrying adjacent host genes; high efficiency due to phage integrase.

• Clinically important: introduction of diphtheria toxin, cholera toxin, Shiga toxin, botulinum toxin genes.

Plasmid Highlights

• Bonus functions: antibiotic synthesis, toxin production, metabolic flexibility, enhanced virulence.

• Major driver of multidrug resistance spread in hospitals via conjugation.

Classical Genetic Engineering Workflow

1. Identify & Amplify the Gene of Interest (GOI)

• Isolate double-stranded DNA containing GOI.

• PCR components

  • DNA primers (more heat-stable than RNA primers).

  • dNTPs: A, T, C, G.

  • Heat-stable DNA polymerase (originally Taq from Thermus aquaticus).

• Thermocycler steps

  1. Denature (high T) – strands separate.

  2. Anneal (cool) – primers bind.

  3. Extend (warm) – polymerase synthesises copy.

• Exponential amplification ⇒ millions of GOI copies.

2. Construct Recombinant Plasmid

• Choose lab plasmid with:

  • Origin of replication

  • Selectable marker (antibiotic resistance)

  • Multiple cloning site (MCS) with unique restriction enzyme sites (e.g. EcoRI $\text{TTAA}$ palindrome).

• Digest plasmid and GOI with same enzyme ⇒ complementary sticky ends.

• Mix; sticky ends base-pair.

• Add DNA ligase to seal backbone ⇒ stable recombinant.

3. Transformation & Plate Selection

• Introduce plasmids into competent E. coli.

• Grow on agar containing the marker antibiotic.

  • Growth = plasmid present.

• Blue/white screen (lacZ interruption)

  • No insert → intact lacZ → cleaves X-gal → blue colonies.

  • Insert present → disrupted lacZ → white colonies.

• Pick white, antibiotic-resistant colony ⇒ clone carries GOI.

4. Plant Engineering via Agrobacterium tumefaciens

• Natural Ti plasmid (transfer plasmid) delivers DNA into plant genome.

• Strategies

  • Stable transgenics: transform single cells, regenerate whole plant with permanent insertion.

  • Transient expression: sand-abrasion + vacuum infiltration of leaves; high-level protein production then plant dies (useful for vaccine/therapeutic manufacturing).

Applications of Recombinant Technology

• Nucleic-acid therapeutics: gene therapy, RNA probes, DNA vaccines.

• Engineered microbes: insulin, growth factors, vaccine antigens, bioremediation.

• Transgenic plants: pest resistance (Bt corn), drought tolerance, edible vaccines, pharma proteins (Ebola antibody in tobacco).

• Transgenic / humanized animals

  • Disease models (e.g. Regeneron’s human-immune-system mice).

  • Limited “pharming” applications due to public concern.

• Xenografting

  • Gene-edited pig organs expressing human surface markers to reduce rejection; experimental heart transplants achieved ~2 weeks survival so far.

Virus Fundamentals

Living vs. Non-living

• No metabolism, motility, or independent replication; obligate intracellular parasites.

• Still biological entities composed of macromolecules.

Minimal Structure

1. Nucleic acid genome – DNA or RNA, single- or double-stranded, linear or circular.

2. Capsid – protective protein shell (built from repeating capsomeres).
   Nucleic acid + capsid = nucleocapsid.

3. Spikes (attachment proteins) – bind specific host receptors.

4. Envelope (in some animal viruses)

   • Host-derived phospholipid bilayer acquired during egress; spikes embed here.

Size Spectrum

• Smallest: Poliovirus ~30 nm.

• Largest animal virus: Poxvirus ~300 nm; still <½ size of E. coli.

Morphologies

• Icosahedral (20-sided) – many DNA and RNA viruses.

• Helical – e.g. influenza, TMV.

• Complex

  • All bacteriophages (icosahedral head + helical tail).

  • Only animal example: Poxviridae (brick-shaped “genome sandwich”).

Host Range, Tropism & Specificity

• Tropism: distribution of receptor molecules differs by tissue & species.

• Specificity levels

  • Species: smallpox 👉 humans only; rabies 👉 most mammals.

  • Tissue: Hepatitis B 👉 hepatocytes; measles 👉 widespread (binds sialic acid, common on many cell types).

• Receptor examples

  • HIV: primary $\text{CD4}$ + coreceptor $\text{CCR5\ or\ CXCR4}$.

  • SARS-CoV-2: ACE2 enzyme (impacts blood-pressure regulation).

  • Many respiratory/GI viruses: Sialic acid.

Bacteriophage Infection Strategies (Parallels to Animal Viruses)

Viral Classification Snapshot (Baltimore Scheme)

• Primary division by genome type.

  • dsDNA, ssDNA, dsRNA, ssRNA(+), ssRNA(–), reverse-transcribing DNA/RNA.

• DNA virus families (examples): Adenoviridae, Herpesviridae, Poxviridae, Hepadnaviridae (HBV), Parvoviridae (ssDNA).

• RNA virus families (examples): Picorna (polio), Orthomyxo (influenza A/B), Paramyxo (measles, RSV), Rhabdo (rabies), Retro (HIV), Reoviridae (dsRNA – rotavirus).

• All negative-strand RNA viruses are enveloped and carry their own RNA-dependent RNA polymerase.

General Animal-Virus Replication Cycle

1. Attachment & Recognition – spike binds complementary receptor; determines host range.

2. Penetration

   • Enveloped: Fusion with plasma membrane.

   • All viruses: Receptor-mediated endocytosis (cell “eats” the virion).

3. Uncoating – capsid removed; genome liberated.

4. Synthesis (Gene Expression & Genome Replication)

   • Early genes: capsid proteins, polymerases, host-subversion factors.

   • DNA viruses usually replicate in nucleus; RNA viruses in cytoplasm (exceptions exist).

5. Assembly – spontaneous self-assembly once components accumulate.

6. Maturation – additional processing (protease trimming, conformational changes) to make particles infectious.

7. Release

   • Budding (most enveloped) – gradual loss of membrane, eventual cell death.

   • Lysis (non-enveloped) – cell ruptures when virion load reaches threshold.

Disease Patterns in Humans

• Acute: rapid onset & clearance (or death).

  • Examples: Influenza, Rhinovirus.

  • Immune system often clears infection or host succumbs (e.g. symptomatic rabies $\to$ 100 % fatal).

• Persistent

  1. Chronic – continuous low-level production; virus detectable.

  • Examples: Hepatitis B, HIV.

  1. Latent – genome silent between reactivations.

  • Examples: HSV-1/2 cold sores; Varicella zoster ($\text{chickenpox} \to \text{shingles}$).

Oncogenic Potential

• Tumor-associated viruses: HPV, HBV, HCV, EBV, HTLV-1, Kaposi’s sarcoma herpesvirus.

• Three mechanisms

  1. Insertional activation: viral integration turns proto-oncogene $\to$ oncogene (HPV).

  2. Chronic inflammation/regeneration: long-term damage drives mutations (HCV-induced hepatocellular carcinoma).

  3. Transduced oncogenes: phage-like mobilization of host oncogene into new cell (theoretical; evidence in genome analyses).

• Overall, viruses account for <20 % of human cancers; majority still arise from spontaneous or environmental mutations.

Ethical, Practical & Real-World Connections

• Negative/positive selection underpin modern antibiotic-susceptibility testing and microbial genetics labs.

• Ames test forms part of EPA & FDA chemical-safety pipelines.

• Conjugation-driven resistance shapes hospital antibiotic-stewardship policies.

• Recombinant DNA tech fuels pharmaceutical manufacturing (insulin, monoclonal antibodies), agriculture (Bt crops), and emerging xenotransplantation.

• Viral host-range insights inform zoonotic surveillance (e.g. SARS-CoV-2 spillover risk) and vaccine-target choice (spike proteins).

• Persistent vs. acute infection paradigms guide antiviral therapy duration and public-health isolation periods.