𧬠Genetic Elements
Chromosome
- Usually one copy-type circular molecule
- Covalently closed
- Often several copies per cell
- Supercoiled into loops around a protein core
- Single-strand nick relaxes only one loop
- About 2,000 genes
- Carries every essential gene
Plasmid
- Extrachromosomal, circular, small (1.5β400 kb)
- Replicates independently
- Carries nonessential genes
- Fertility (tra) genes
- Antibiotic resistance genes
- Most exotoxin genes
- Episome = plasmid able to integrate into the chromosome
- Needs matching insertion sequences
Prophage
- Repressed temperate phage DNA inside the chromosome
- May encode virulence proteins
- Virulence gain by prophage = lysogenic conversion
High-yield
- Essential genes live on the chromosome, never on plasmids.
- Toxin and resistance genes commonly ride on plasmids or prophages.
π Recombination Types
Why recombination is needed
- Binary fission gives identical daughters
- Only de novo mutation changes them
- Incoming DNA must be stabilized
- Linear fragment (exogenote) is degraded by exonucleases
- Stabilized by integration or by re-forming a circular plasmid
| Feature | Homologous | Site-specific |
|---|---|---|
| Sequence match | Extensive homology required | Only a short attachment site |
| Outcome | Exchange of segments | Whole molecule inserted (sum of both) |
| Key factors | recA (essential), recB, recC, recD | Specific integration enzymes at sites |
| Fate of leftover | Displaced linear piece is lost | Nothing lost |
| Examples | Stabilizing transformed or transduced DNA | Hfr formation, prophage insertion, transposition |
Exam trap
- recA-deficient cells cannot stabilize linear exogenotes.
- Transposition is a form of site-specific integration.
𧫠Transformation & Conjugation
Transformation Uptake of naked DNA
Cell diesβFree DNA releasedβCompetent cell binds fragmentβUptakeβrecA integration
- Naturally competent genera
- Haemophilus influenzae
- Streptococcus pneumoniae
- Bacillus
- Neisseria
- Classic outcome: rough, unencapsulated pneumococcus becomes smooth and virulent
Conjugation Direct contact, one strand moves
- Donor carries fertility (F) factor
- tra region: sex pilus, pair stabilization, DNA transfer genes
- oriT: site of the nick where transfer starts
- Recipient (Fβ) lacks the F factor
- Transfer is one-way, donor to recipient
- Donor copy is re-synthesized, so donor genotype is unchanged
| Cross | F factor state | What moves | Recipient result |
|---|---|---|---|
| F+ Γ Fβ | Free plasmid | Whole plasmid (oriT first, tra last) | Becomes F+; no chromosomal genes; no recombination needed |
| Hfr Γ Fβ | Integrated (episome) | oriT, part of F, then adjacent chromosome genes | Stays Fβ; gains a few chromosomal genes; needs recombination |
Hfr details
- Genes nearest the integration site transfer most often
- Full chromosome transfer takes about 2 hours
- Mating bridge usually breaks early
- tra end is almost never transferred
- So recipient does not become Hfr
High-yield
- F+ Γ Fβ converts the recipient; Hfr Γ Fβ does not.
- Gene transfer order maps linear gene position.
π¦ Phages & Transduction
Virulent
- Lytic cycle only
- Attach β inject DNA β make early proteins β copy DNA β make structural proteins β assemble β lyse
Temperate
- Can choose lysis or lysogeny
- Repressor wins early β prophage
- Repressor loss β lytic cycle
Lysogeny
- Prophage copies with each host division
- Persists while repressor is functional
- Defective prophage needs a helper phage to replicate
- Lambda phage in E. coli
- Single insertion site, between gal and bio
- att sites mediate integration
- Model for retroviral provirus
Induction
UV / alkylating damageβRepressor damagedβProphage excisedβLytic cycle
- Usually precise excision gives normal phage
- Rare imprecise excision gives a transducing phage
Transduction types
| Generalized | Specialized | |
|---|---|---|
| Phage type | Lytic cycle (virulent or temperate) | Temperate, lysogenic |
| Error | Bacterial DNA packaged by mistake | Faulty excision takes neighboring genes |
| Genes moved | Any bacterial gene | Only genes beside the insertion site (gal or bio for lambda) |
| Prophage needed | No | Yes |
| Stabilization | Homologous recombination | Homologous recombination |
Phage-linked virulence (lysogenic conversion)
| Bacterium | Phage-encoded factor |
|---|---|
| Corynebacterium diphtheriae | Diphtheria toxin |
| Clostridium botulinum | Botulinum toxin |
| Vibrio cholerae | Cholera toxin |
| Streptococcus pyogenes | Pyrogenic (erythrogenic) toxins |
| E. coli (EHEC) / Shigella | Shiga-type toxin |
| Salmonella | Altered O antigen |
βοΈ Transfer Mechanisms Compared
| Question | Transformation | Conjugation | Transduction |
|---|---|---|---|
| Cell contact needed | No | Yes | No |
| Phage involved | No | No | Yes |
| Competence needed | Yes | No | No |
| Naked DNA | Yes | No | No |
| Recombination to stabilize | Yes | F+: no; Hfr: yes | Yes |
Exam traps
- Only conjugation needs pili and oriT.
- Only transduction needs a phage vector.
- Only transformation needs free DNA and competence.
π Drug Resistance
| Type | Genetic location | Typical mechanism | Level |
|---|---|---|---|
| Intrinsic | Inherent biology | Target absent or unreachable | β |
| Chromosomal | Chromosome | Altered drug target | Usually low |
| Plasmid-mediated | R plasmid | Drug-modifying enzymes, efflux, target change | Often high; transferable |
Intrinsic examples
- No mycolic acid β isoniazid ineffective
- No peptidoglycan (Mycoplasma) β penicillin ineffective
R plasmids
- R factor = RTF + R determinant
- RTF: oriT and tra genes, conjugation machinery
- R determinant: resistance genes
- Multidrug plasmids assemble through transposons and gene cassettes
- Mobile DNA, encodes transposase
- Insertion can disrupt unrelated genes
- Found in bacteria, eukaryotes, viruses
Mechanisms by drug class
| Drug | Resistance mechanism |
|---|---|
| Penicillins, cephalosporins | Ξ²-lactamase cleaves ring (plasmid); altered PBPs (chromosome) |
| Methicillin (MRSA) | Mutated penicillin-binding protein |
| Aminoglycosides | Acetyl-, adenylyl-, phosphotransferases; reduced uptake |
| Chloramphenicol | Acetyltransferase |
| Tetracyclines | Efflux; ribosome-protecting protein |
| Sulfonamides | Export; low-affinity enzyme |
| Vancomycin | Cell wall precursor ends in D-AlaβD-lactate (vanA, vanB); thicker wall in VISA |
| Macrolides, lincosamides | 23S rRNA methylation; efflux; esterases |
| Fluoroquinolones | Mutant topoisomerase; efflux |
| Rifampin | Mutant RNA polymerase |
| Isoniazid | Loss of catalase-peroxidase activation |
| Ethambutol | Mutant arabinosyl transferase |
| Trimethoprim / sulfonamide | Altered DHFR / altered dihydropteroate synthase |
High-yield
- vanA and vanB moved from Enterococcus to Staphylococcus aureus (VRSA).
- ESBLs can be missed by routine susceptibility tests.
- Gram-negative Ξ²-lactamases act in the periplasm.
π¬ Susceptibility Testing
Disk diffusion
Lawn of isolateβDrug disks placedβDiffusion gradientβMeasure inhibition zone
- Result: susceptible, intermediate, or resistant
- Pros: cheap, simple, many drugs per plate
- Con: qualitative only
MIC vs MBC
| MIC | MBC | |
|---|---|---|
| Measures | Growth inhibition | Killing |
| Method | Serial drug dilutions plus fixed inoculum | Subculture MIC tubes onto drug-free agar |
| Endpoint | Lowest concentration with no visible growth | Lowest concentration with no regrowth |
| Control | Drug-free tube confirms viable inoculum | β |
| Use | Routine | Ordered selectively |
- MBC matters when host immunity cannot clear inhibited bacteria
- Immunocompromised patients
- MBC is equal to or above MIC
Exam trap
- A lower MIC does not by itself prove bactericidal action.
β Rapid Review
Conjugation
- Needs pilus and oriT
- Donor genotype unchanged
- F+ cross makes new F+
- Hfr cross leaves Fβ
Transformation
- Free DNA, competent cells
- Pneumococcus, Haemophilus, Neisseria, Bacillus
Transduction
- Phage is the vehicle
- Generalized: any gene
- Specialized: neighbors of prophage
Resistance
- Plasmids spread it horizontally
- Transposons assemble multidrug plasmids
- Chromosomal changes mostly alter targets
Clinical pearl
- Phage-encoded toxins explain why only some strains of a species cause toxin disease.